Gene cluster

ABSTRACT

Novel isolated DNA sequences which comprise all or part of a gene cluster encoding sanglifehrin synthase, processing and regulatory genes involved in the biosynthesis of a mixed non-ribosomal peptide/polyketide compound, or mutants having altered biosynthetic capability, polypeptides or mutants thereof encoded by DNA or the mutants, vectors containing the DNA or the mutants thereof, host cells transformed with the DNA, the mutants thereof, or the vector, and a method for producing sanglifehrin compounds. Compounds with cyclophilin inhibition activity used as immunosuppressants, antivirals or cardiac protection agents.

The present application is §371 application of PCT/CN2009/074178 filed Sep. 24, 2009, which claims priority to CN Application No. 200810200388.4, filed Sep. 24, 2008. The entire disclosure of each of the foregoing applications is incorporated by reference herein.

INTRODUCTION

The present invention relates to novel isolated DNA sequences which comprises a gene cluster encoding sanglifehrin synthase, processing and regulatory genes involved in the biosynthesis of a mixed non-ribosomal peptide/polyketide compound, or mutants having altered biosynthetic capability, polypeptides or mutants thereof encoded by DNA or the mutants, vectors containing the DNA or the mutants thereof, host cells transformed with the DNA, the mutants thereof, or the vector, and a method for producing sanglifehrin compounds. The present invention also provides compounds with cyclophilin inhibition activity useful as immunosuppressants, antivirals or cardiac protection agents.

FIELD OF THE INVENTION

The invention relates to the field of microorganism genetic resources and genetic engineering, and in particular relates to the cloning, sequence analysis, in vivo functional verification, and the use of the gene cluster for the biosynthesis of the immunosuppressant sanglifehrin A and related analogues.

BACKGROUND OF THE INVENTION

The immunosuppressant sanglifehrin A (SFA) is a mixed polyketide-peptide natural product from Streptomyces sp. A92-308110, also known as Streptomyces flaveolus or Streptomyces sp. DSM 9954—these titles are all used interchangeably in this and related documents (Sanglier et al., 1999; Fehr et al., 1999; WO 97/02285). Isolation of more than twenty structural analogues of sanglifehrin have been published to date, and SFA has one of the highest immunosuppressant activities among these analogues (Kallen et al., 2005; Sanglier et al., 1999). In SFA, the 22-membered macrolide backbone consists structurally of a polyketide carbon chain and a tripeptide chain. The peptide chain comprises one natural amino acid: valine, and two non-natural amino acids: (S)-m-tyrosine and (S)-piperazic acid, linked by an amide bond, and it is the β-nitrogen atom at position 1 of piperazic acid that is involved in amide bond formation, which stands in contrast to all other piperazic acid containing natural products isolated so far. In addition, a spirocyclic unit is linked to the macrolide by a polyketide long chain, forming a basket structure. The spirocyclic moiety contains nine chiral centers (SFA has seventeen in total) with a quaternary carbon center in the middle, which is unique in currently described natural products. A series of analogues have been directly isolated from fermentation broths of Streptomyces sp. A92-308110 (S. flaveolus), including sanglifehrin B, C, D, E, F, G, H, I, J, K and L (Fehr et al., 1999, Sanglier et al., 1999, WO98/07743). Sanglifehrin B (SFB), in particular, has been shown to possess higher immunosuppressive activity than SFA in MLR assays (Sanglier et al., 1999). Though the total synthesis of SFA and its macrocyclic analogues were achieved with heroic efforts (Sedrani et al., 2003; Nicolau et al., 1999; Paquette et al., 2002; Metternich et al., 1999), no specific in vivo and in vitro studies have been carried out on its biosynthetic pathway.

SFA has strong immunosuppressive activity (Powell and Zheng, 2006), inhibits HIV and HCV infection (Zander et al., 2003; Sokolskaja et al., 2004; Watashi et al., 2005) and prevents severe cardiac cell death caused by the pathological opening of mitochondrial membrane permeability transition pore (MPTP; Clarke et al., 2002). Compared with the immunosuppressants currently in clinical use, such as cyclosporin A (CsA), FK506 and rapamycin, SFA has a similar functional mechanism while having a different target effector site (Hartel et al 2006, Zhang & Liu., 2001; Zenke et al., 2001). CsA binds to cyclophilin A (CypA) (Handschumacher et al., 1984), whilst FK506 and rapamycin bind to FKBP, to form complexes (Schreiber, 1991); the CsA-CypA and FK506-FKBP complexes interact with the same target protein, calcineurin, thereby inhibiting the serine/threonine phosphatase activity of calcineurin, and blocking the production of cytokines, especially the transcription of interleukin 2 (IL-2), which finally lead to T cell arrest in G₀-G₁ stage (Liu et al., 1991). Rap-FKBP complexes interact with the protein kinase FRAP (also known as RAFT or mTOR) (Brown et al., 1994), and prevent phosphorylation of the IL-2 receptor on T cells, leading to arrest of the growing of T cells in G1-S stage. Whilst SFA has been shown to bind to cyclophilins such as Cyclophilin A and B, and inhibit their isomerase activities (Zenke et al 2001), currently the effector protein for SFA-CypA complex remains unknown.

Since the effector protein of the SFA-CypA complex has not yet been found, it was suggested that the immunosuppressive activity of SFA is not mediated directly via the the SFA-CypA complex. In the past 3 years, studies from many scientific groups have shown that SFA can competitively prevent NF-κB from binding the transcription site upstream of the P53 gene, to activate P53 and further inhibit the downstream Cyclin E-cdk2 phosphorylation of the signal pathway, thereby inhibiting the high phosphorylation of Rb in response to IL-2, and making cells insensitive to to IL-2, which forces them to remain in the G1-S stage (Zhang & Liu, 2001). Secondly, by an unknown mechanism, SFA inhibits production of IL-12p⁷⁰ while not affecting the growth of human dendritic cells (Steinschulte et al., 2003). IL-12p⁷⁰ plays a key role in regulating proliferation of Th1 and NK cells, and is the bridge linking innate immunity with adaptive immunity. In addition, the immunosuppressive drugs that are commercially available can lead to severe renal and central nervous system toxicity side effects (Paquette et al., 2002), thus their uses in some immune dysfunction diseases are hindered (for example, calcineurin is the underlying cause of both immunosuppressive and toxic effects of CsA and FK506). With the aim of developing an alternative immunosuppressant or immune modifier, other groups have carried out some development of SFA as a new generation of potent immunosuppressant with lower toxicity (WO 97/02285).

The study of structure-activity relationships between SFA macrocyclic fragments and CypA by X-ray diffraction showed that the tripeptide structure is embedded in the groove of CypA and is important for binding, while the side chain hydroxy group and carbonyl group respectively at positions 17 and 14 are not critical for binding; removal of the trans-diene from the saturated region C18-C22 reduces the binding constant 7 fold, suggesting the trans-diene stabilizes the conformation (Sedrani et al., 2003). A computer-modeling study shows that the spirocyclic unit of SFA may also contribute to the stability of the SFA-CypA binding (Pemberton et al., 2003). The crystal structure of the complete SFA-CypA complex shows that the binding regions in SFA-CypA are substantially the same as those in CsA-CypA, and both SFA and CsA mainly interact with residue W121, R55, H126, N102 and Q63; the C24-C32 chain between the macrocycle and the spirocyclic moiety make van der Waals contacts with residues I57, T119 and W121 of CypA; in addition, the presence of the long polyketide chain of SFA imposes a side-chain reorientation on W121, as compared with the crystal structure of CypA; within the spirocycle, only the methyl group C45 makes vdW contacts with side-chain atoms from I57 and F60 of CypA (Kallen et al., 2005).

The SFA-CypA complex can exist in a stable dimeric form, as shown by gel filtration chromatography. Based on crystal analysis, with the exception of the spirobicyclic and α-ketobutyrate moieties, all of the remaining parts of SFA are deeply buried in the dimer; the E,E-diene region C18-C22 is not involved in direct contacts with the CypA but instead forms vdW contacts with the meta-tyrosine of neighboring SFA within the dimer, which favors the dimeric association in the complex; the two SFA molecules make vdW contacts with each other in the region C18-C22; and a direct hydrogen bond links W121 of one CypA molecule and R148 of another CypA molecule in the dimer complex.

Using the streptomycete that is known to produce SFA, Streptomyces sp. A92-308110 (S. flaveolus), the inventors of the present invention cloned the biosynthetic gene cluster thereof, and further studied the biosynthesis of SFA by methods combining microbiology, molecular biology, biochemistry and organic chemistry. Through study of the biosynthesis, the enzymatic mechanism which generates distinctive chemical structures such as piperazic acid was revealed. Based on this, genetic modifications were made to the SFA biosynthetic pathway, and novel compounds were produced.

The present invention is particularly useful as it should enable the commercial application of recombinant DNA technology and biosynthetic engineering to increase the yield of sanglifehrins and generation of novel sanglifehrin analogues.

SUMMARY OF THE INVENTION

The present invention advantageously provides novel DNA sequences and proteins involved in the production of biosynthetic gene products, in particular biosynthesis of sanglifehrin. Specific embodiments of the genes and proteins are detailed in the accompanying sequence listing and the following description. SEQ ID No. 1 provides the nucleic acid responsible for the biosynthesis of sanglifehrin A.

Thus, the invention relates to the cloning, sequence analysis, functional verification, in vitro biochemical analysis and the use of the biosynthetic gene cluster of SFA, an immunosuppressive polyketide-nonribosomal peptide natural product produced by Streptomyces sp. A92-308110 (S. flaveolus) (available from the DSMZ, Braunschweig, Germany as Streptomyces sp. DSM 9954). In addition, targeted alterations were made to the genes encoding the biosynthetic pathway for generation of sanglifehrin, leading to microbial strains producing novel sanglifehrin analogues.

The invention allows direct manipulation of sanglifehrin A and related chemical structures via biosynthetic engineering of the genes and proteins involved in the biosynthesis of the sanglifehrin A. These chemical modifications may be either impossible or unfeasible to do by chemical methodologies due to the complexity of the structures.

The gene cluster isolated and characterized in this way enables targeted optimization of the production of sanglifehrin and sanglifehrin analogues, for example, by duplication of the gene cluster, or parts of the cluster, by overexpression of genes (in particular positive regulatory genes) using plasmid vectors and non-natural promoters, or by inactivation of negative regulators.

In addition, the sequenced and characterized cluster enables targeted biosynthetic preparation of sanglifehrin analogues, a number of examples of which are included in this document.

Examples include the following:

-   -   Inactivation of genes coding for proteins involved in individual         biosynthetic steps, for example by gene disruption (see e.g. WO         2004/007709; WO 2004/058976).     -   Replacement of genes coding for proteins involved in individual         biosynthetic steps, by gene replacement or by disruption         followed by separate expression of genes from other biosynthetic         pathways (see e.g. Gaisser et al., 2001; WO 01/79520; WO         2005/054266; WO2005/054265)     -   Exchange of individual modules or domains within the polyketide         synthase (“PKS”) or non-ribosomal peptide synthase (“NRPS”) with         modules or domains from other PKS or NRPS clusters, to enable         generation of novel sanglifehrin analogues (for example as         described in Oliynyk et al., 1996; WO 98/01546; WO 00/01827;         Staunton and Wilkinson, 2001; Sheehan et al., 2006)     -   Use of the gene sequence to identify related biosynthetic         clusters from other organisms, for example by use as a DNA probe         (see e.g. Shen et al., 2002; Liu et al., 2002; Li et al., 2004;         Huang et al., 2005; Jia et al., 2006; Fang et al., 2008).

Thus according to a first aspect of the invention there is provided an isolated nucleic molecule comprising:

-   -   (a) the sanglifehrin A biosynthesis gene cluster nucleic acid         comprising SEQ ID No. 1;     -   (b) a nucleic acid having at least 80% sequence identity (e.g.         at least 85 or 90 or 95 or 96 or 97 or 98 or 99% sequence         identity) to the nucleic acid of (a) and which codes for         polypeptides having the same enzymatic and regulatory activities         for making a polyketide or a polyketide starter unit as those         encoded by the nucleic acid of (a);     -   (c) a nucleic acid encoding one or more polypeptides having at         least 80% amino acid sequence identity (e.g. at least 85 or 90         or 95 or 96 or 97 or 98 or 99% sequence identity) to one or more         polypeptides encoded by the nucleic acid of (a) and which codes         for one or more polypeptides having one or more of the necessary         enzymatic or regulatory activities for making a polyketide or a         polyketide starter unit or precursor;     -   (d) a nucleic acid portion of (a), (b) or (c) which codes for a         polyketide synthase or a non-ribosomal peptide synthase         polypeptide or an enzymatically active module of either, or a         polyketide starter unit or precursor biosynthesis gene product         or a polyketide biosynthesis regulatory polypeptide; or     -   (e) a nucleic acid portion of (d) which encodes an enzymatically         active domain of a polyketide synthase or a non-ribosomal         peptide synthase polypeptide or an enzymatically active module         of either, or a polyketide starter unit or precursor         biosynthesis gene product or a polyketide biosynthesis         regulatory polypeptide.

This and other aspects of the invention will be elaborated in the foregoing disclosure.

Definitions

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example “an analogue” means one analogue or more than one analogue.

As used herein the term “analogue(s)” refers to chemical compounds that are structurally similar to another but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group).

As used herein the term “polyketide” refers to any molecule generated via biosynthesis involving a polyketide synthase (PKS). This may additionally include some elements from non-ribosomal peptide synthase (NRPS) domains and/or further biosynthetic modification, such as methylation or hydroxylation.

As used herein the term “hybrid polyketide” refers to any molecule generated via biosynthesis involving a polyketide synthase (PKS), where the gene cluster encoding this polyketide synthase has been altered by human intervention to lead to a different biosynthetic product. This may additionally include some elements from non-ribosomal peptide synthase domains and/or further biosynthetic modification, such as methylation or hydroxylation. The alterations themselves may include, but are not limited to, site directed mutagenesis of domains (eg acyltransferase domains), replacement of domains, modules or genes from the same or a heterologous PKS or NRPS cluster.

As used herein the term “high stringency conditions” means conditions where only very closely related or identical DNA sequences are hybridized. This is frequently done in Southern hybridisation by increasing the temperature of the wash buffer. For oligonucleotide probes the hybridization step can be performed at 5° C. above T_(m) for perfectly matched sequences, where T_(m) is calculated using a formula such as T_(m)=4×(number of GC base pairs)+2×(number of AT base pairs). An example of high stringency conditions is given in the section below entitled “Nucleic acid hybridization”.

As used herein the term “heterologous host” in relation to a nucleic acid sequence, particularly a sanglifehrin biosynthetic cluster or part thereof, means a host that would not naturally contain such a nucleic acid sequence.

As used herein the term “heterologous” in connection with, for example a domain or module of a sanglifehrin PKS or NRPS, means a domain or module that would not naturally be present in that PKS or NRPS.

As used herein the term “non-native” means in connection with, for example, a domain or module of a sanglifehrin PKS or NRPS, a domain or module that would not naturally be present in that location in that PKS or NRPS; for instance it might be heterologous (i.e. from a different PKS or NRPS) or else it might be present in a different location in the same PKS or NRPS.

The pharmaceutically acceptable salts of compounds of the invention such as the compounds of formula (I) include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases as well as quaternary ammonium acid addition salts. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic and the like. Hydrochloric acid salts are of particular interest. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts. More specific examples of suitable basic salts include sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts. References hereinafter to a compound according to the invention include both compounds of formula (I) and their pharmaceutically acceptable salts.

Alkyl, alkenyl and alkynyl groups may be straight chain or branched.

Examples of alkyl e.g. C1-C4 alkyl groups include methyl, ethyl, n-propyl, i-propyl and n-butyl.

DETAILED DESCRIPTION OF THE INVENTION

The whole gene cluster according to the invention comprises the nucleotide sequences or complementary sequences of 24 genes (SEQ ID NO: 1), including:

-   -   one non-ribosomal peptide synthetase (NRPS) gene, sfaD,         comprising altogether 3 modules, 10 functional domains, and         responsible for the formation of the peptide moiety of the         macrocyclic backbone;     -   five linear polyketide synthase (PKS) genes, sfaE, sfaF, sfaG,         sfaH, sfaI, responsible for the formation of the spirocycle, the         polyketide long chain and the polyketide moiety of the         macrocyclic backbone;     -   one iterative polyketide synthase gene, sfaK, comprising 4         functional domains, and responsible for the biosynthesis of a         special 6-carbon precursor en route for biosynthesis of the         unusual 7-carbon extension unit;     -   Three genes, sfaA, sfaB, sfaJ responsible for the biosynthesis         of non-natural amino acid precursor building blocks;     -   Seven genes sfaM, sfaN, sfaP, sfaQ, sfaL, sfaR, sfaO,         responsible for biosynthesis of precursors, such as those for         the starter unit;     -   one regulatory gene, sfaC, associated with the fermentation         yield of SFA;     -   one MbtH protein encoding gene, sfaS, postulated to be         associated with the regulation of the NRPS; and     -   five genes with unknown function, sfaU1, sfaU2, sfaV1, sfaV2,         sfaV3.

Thus the invention also provides an isolated nucleic molecule comprising one or more of (a) a linear PKS gene selected from sfaE, sfaF, sfaG, sfaH and sfaI, or (b) a NPRS gene sfaD, or (c) an iterative PKS gene sfaK, or (d) a starter unit or precursor biosynthesis gene selected from sfaA, sfaB, sfaJ, sfaM, sfaN, sfaP, sfaQ, sfaL, sfaO, or (e) a regulatory gene sfaC, or (f) a MtbH protein encoding gene sfaS, or (g) or a crotonyl-CoA reductase gene sfaR, or (h) a gene of unknown function selected from sfaU1, sfaU2, sfaV1, sfaV2, and sfaV3.

The aforementioned genes are typically defined by nucleic acids encoding for the proteins of SEQ ID Nos. 2-25.

In particular, the invention provides an isolated nucleic molecule comprising one or more of (a) a linear PKS gene selected from sfaE being residues 30707-37360 of SEQ ID No. 1, sfaF being residues 37394-50014 of SEQ ID No. 1, sfaG being residues 50017-60903 of SEQ ID No. 1, sfaH being residues 60918-85823 of SEQ ID No. 1 and sfaI being residues 85823-96040 of SEQ ID No. 1 or (b) a NRPS gene sfaD being residues 19885-30714 of SEQ ID No: 1 or (c) an iterative PKS gene sfaK being residues 97396-101943 of SEQ ID No. 1, or (d) a starter unit or precursor biosynthesis gene selected from sfaA being residues 17024-17854 of SEQ ID No. 1, sfaB being residues 17851-19191 of SEQ ID No. 1, sfaJ being residues 96225-97391 of SEQ ID No. 1, sfaM being residues 103210-103929 of SEQ ID No. 1, sfaN being residues 104001-105023 of SEQ ID No. 1, sfaP being residues 105366-107216 of SEQ ID No. 1, sfaQ being residues 107366-108145 of SEQ ID No. 1, sfaL being residues 101936-103213 of SEQ ID No. 1, sfaO being residues 105091-105345 of SEQ ID No. 1, or (e) a regulatory gene sfaC being residues 19193-19888 of SEQ ID No. 1, or (f) a MtbH protein encoding gene sfaS being residues 109583-109798 of SEQ ID No. 1, or (g) or a crotonyl-CoA reductase gene sfaR being residues 108150-109511 of SEQ ID No. 1, or (h) a gene of unknown function selected from sfaU1 being residues 14973-15413 of SEQ ID No. 1, sfaU2 being residues 15596-16063 of SEQ ID No. 1, sfaV1 being residues 109776-110312 of SEQ ID No. 1, sfaV2 being residues 111285-111743 of SEQ ID No. 1 and sfaV3 being residues 112218-112652 of SEQ ID No. 1; or comprising a nucleic acid sequence which encodes one or more of the same polypeptides as those encoded by the aforementioned genes but which differ only by virtue of the redundancy of the genetic code; or comprising a nucleic acid sequence capable of hybridizing to one or more of the above gene sequences under conditions of high stringency; or comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to one or more of the above gene sequences and which encodes a polypeptide having the same function as the corresponding gene product; or comprising a nucleic acid sequence encoding one or more polypeptides having at least 80% amino acid sequence identity (e.g. at least 85 or 90 or 95 or 96 or 97 or 98 or 99% sequence identity) to one or more of the polypeptides encoded by the aforementioned genes and which have the same function; or fragments comprising at least 50, 100, 200, or 500 consecutive nucleotides of one or more of the aforementioned genes; or the complement of any of the aforementioned nucleic acid sequences.

There is also provided an isolated nucleic molecule comprising one or more of (a) a module or domain from a PKS gene selected from sfaE being residues 30707-37360 of SEQ ID No. 1, sfaF being residues 37394-50014 of SEQ ID No. 1, sfaG being residues 50017-60903 of SEQ ID No. 1, sfaH being residues 60918-85823 of SEQ ID No. 1 or sfaI being residues 85823-96040 of SEQ ID No. 1 or (b) a module or domain from the NRPS gene sfaD being residues 19885-30714 of SEQ ID No: 1, or (c) a domain from the iterative PKS gene sfaK being residues 97396-101943 of SEQ ID No. 1; or comprising a nucleic acid sequence which encodes one or more modules or domains of the same polypeptides as those encoded by the aforementioned genes but which differ only by virtue of the redundancy of the genetic code; or comprising a nucleic acid sequence capable of hybridizing to one or more of the above nucleic acid molecules under conditions of high stringency; or comprising a nucleic acid sequence having at least 70% identity to one or more of the above nucleic acid molecules and which encodes a polypeptide having the same function as a module or domain of the corresponding gene product; or comprising a nucleic acid sequence encoding one or more polypeptides having at least 80% amino acid sequence identity to one or more of the polypeptides encoded by a module or domain of the aforementioned genes and which have the same function; or fragments comprising at least 50 consecutive nucleotides of one or more of the modules or domains of the aforementioned genes; or the complement of any of the aforementioned nucleic acid sequences.

A module or domain of a gene is the portion of the gene that encodes a module or domain of the corresponding gene product.

There is also provided a hybrid polyketide producing gene cluster nucleic acid based on SEQ ID No. 1 in which one or more (e.g. one) domains, modules or genes have been deleted, mutated so as to make inactive or less active an enzymatic or regulatory function, mutated so as to have altered functionality, or replaced by an replacement, or in which one or more (e.g. one) non-native domains, modules or genes have been inserted, for example wherein one or more (e.g. one) domains, modules or genes have been replaced by (a) a domain, module or gene from elsewhere in the sanglifehrin A biosynthesis gene cluster or (b) a domain, module or gene which is heterologous to the sanglifehrin A biosynthesis gene cluster or wherein one or more (e.g. one) domains, modules or genes have been mutated to inactivate or make less active an enzymatic or regulatory function.

For example, there is provided a hybrid polyketide producing gene cluster nucleic acid wherein one or more PKS genes selected from sfaE, F, G, H and I have been modified whereby one or more domains or modules have been deleted, mutated so as to make inactive or less active an enzymatic function or so as have altered functionality or replaced by an replacement or whereby one or more non-native domains or modules have been inserted e.g. whereby one or more domains or modules from elsewhere in the SFA biosynthetic gene cluster or from a heterologous polyketide biosynthetic gene cluster have been inserted.

The modules of the PKS genes sfaE, F, G, H and I may be seen by reference to FIG. 7. Thus the modules may typically comprise KS-AT-ACP or KS-AT-DH-KR-ACP or KS-AT-KR-ACP or KS-AT-DH-ER-KR-ACP domains.

For example:

-   -   an AT domain may be replaced with an AT domain from a         heterologous PKS or from elsewhere within the SFA PKS which has         a different substrate specificity; and/or     -   a DH domain may be deleted or rendered inactive; and/or     -   a DH domain from the SFA PKS or from a heterologous PKS may be         inserted into a module;     -   an ER domain may be deleted or rendered inactive; and/or     -   an ER domain from the SFA PKS or from a heterologous PKS may be         inserted into a module.     -   the reductive loop of a given module (meaning the DH-KR or         DH-ER-KR or the KR domains where present) may be replaced by a         reductive loop having different elements.

In one embodiment, the AT domain of module 13 may be replaced with an AT domain from the SFA PKS or from a heterologous PKS which has a different substrate specificity e.g. one having specificity for methyl malonyl. This particular mutation leads to introduction of a methyl group at position 14.

In one embodiment the DH domain of one or more of modules 1, 3, 6, 7, 8, 10, 11 and 13 is deleted or rendered inactive. These particular mutations lead to introduction of one or more hydroxyl groups into the molecule, for example at positions 21 and/or 25.

There is also provided a hybrid polyketide producing gene cluster nucleic acid wherein the NRPS gene sfaD has been modified whereby one or more domains or modules have been deleted, mutated so as to make inactive or less active an enzymatic function or so as have altered functionality or replaced by an replacement or whereby one or more non-native domains or modules have been inserted.

There is also provided a hybrid polyketide producing gene cluster nucleic acid wherein regulatory gene sfaC has been modified so as to increase or decrease its activity, or deleted.

There is also provided a hybrid polyketide producing gene cluster nucleic acid wherein the iterative PKS gene sfaK has been modified whereby one or more domains have been deleted, mutated so as to make inactive or less active an enzymatic function or so as have altered functionality or replaced by an replacement or whereby one or more non-native domains have been inserted e.g. whereby one or more domains from elsewhere in the SFA biosynthetic gene cluster or from a heterologous polyketide biosynthetic cluster have been inserted.

The module of the iterative PKS gene sfaK may be seen by reference to FIG. 7. Thus the module comprises KS-AT-ACP-KR domains.

There is also provided a hybrid polyketide producing gene cluster nucleic acid wherein one or more starter unit or precursor biosynthesis genes selected from sfaA, B, J, M, N, P, Q, L and O have been deleted or modified so as to decrease their activity or modified or replaced so as to alter their substrate selectivity.

There is also provided a hybrid polyketide producing gene cluster nucleic acid wherein one or more starter unit or precursor biosynthesis genes or one or more operons containing one or more starter unit or precursor biosynthesis genes have been deleted or mutated so as to be inactive or less active at producing said starter unit or precursor than the native gene or operon.

More preferred embodiments of this aspect of the invention include isolated nucleic acid that encodes a domain of the PKS or NRPS of SEQ ID No. 1, residues 19885-30714, 30707-37360, 37394-50014, 50017-60903, 60918-85823, 85823-96040. These nucleic acids can be used, alone or in combination with nucleic acids encoding other PKS or NRPS domains or modules as intermediates, for example in the construction of recombinant vectors.

The present invention also provides for a method for identifying, isolating and cloning nucleic acid including any one of the DNA fragments described above. A preferred method comprises, for example, the following steps:

-   -   a) Setting up a genomic DNA bank (for example a cosmid library)     -   b) Screening this bank with the assistance of the DNA sequences         of this invention     -   c) Isolating the clones identified as positive

A general method for identifying the DNA fragments involved in the biosynthesis of sanglifehrins comprises, for example, the following steps:

-   -   a. Isolating DNA fragments with homology to the sanglifehrin         gene cluster can be carried out by Southern blotting a cosmid         library, probing with DNA fragments (for example of ˜1 kb) from         SEQ ID No. 1 to find cloned fragments with homology to the         sanglifehrin cluster.     -   b. The cosmids which are seen to hybridise to the probe can then         be removed, and the DNA sequenced.     -   c. Adjacent DNA regions can then be isolated by probing the         cosmid library with the labeled cosmid isolated above, to         cosmids containing overlapping DNA.

Other methods are described in Maniatis et al., 1998, Sambrook and Russell, 2001 and Kieser et al., 1999.

The invention further provides a nucleotide sequence, encoding a protein of unknown function. The amino acid sequence it codes for is as shown in SEQ ID NO: 2, and designated as sfaU1, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 14973 to base position 15413.

The invention further provides a nucleotide sequence encoding another protein of unknown function. The amino acid sequence it codes for is as shown in SEQ ID NO: 3, and designated as sfaU2, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 15596 to base position 16063.

The invention further provides a nucleotide sequence encoding a phenylalanine meta-hydroxylase. The amino acid sequence it codes for is as shown in SEQ ID NO: 4, and designated as sfaA, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 17024 to base position 17854.

The invention further provides a nucleotide sequence encoding an ornithine N5-oxygenase. The amino acid sequence it codes for is as shown in SEQ ID NO: 5, and designated as sfaB, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 17851 to base position 19191.

The invention further provides a nucleotide sequence encoding a transcription regulatory factor. The amino acid sequence it codes for is as shown in SEQ ID NO: 6, and designated as sfaC, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 19193 to base position 19888.

The invention further provides a nucleotide sequence encoding a non-ribosomal peptide synthetase comprising the functional domains C, A, PCP, C, A, PCP, C, A, PCP, C, and responsible for biosynthesis of the peptide moiety of the macrocyclic backbone. The amino acid sequence it codes for is as shown in SEQ ID NO: 7, and designated as sfaD, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 19885 to base position 30714.

The invention further provides a nucleotide sequence encoding a polyketide synthase comprising the functional domains ACP, KS, AT, DH, ER, KR, ACP, and responsible for biosynthesis of the precursor of the spirocyclic moiety. The amino acid sequence it codes for is as shown in SEQ ID NO: 8, and designated as sfaE, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 30707 to base position 37360.

The invention further provides a loading domain specific for initiating PKS biosynthesis with a 2-ethylmalonamyl-S-thioester substrate, consisting of the first ACP from sfaE, the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 30707 to base position 31082.

The invention further provides a nucleotide sequence encoding a polyketide synthase comprising the functional domains KS, AT, ACP, KS, AT, KR, ACP, KS, AT, KR, ACP, and responsible for biosynthesis of polyketide long chain. The amino acid sequence it codes for is as shown in SEQ ID NO: 9, designated as sfaF, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 37394 to base position 50014.

The invention further provides a nucleotide sequence encoding a polyketide synthase comprising the functional domains KS, AT, KR, ACP, KS, AT, DH, ER, KR, ACP, and responsible for biosynthesis of much of the polyketide long chain. The amino acid sequence it codes for is as shown in SEQ ID NO: 10, designated as sfaG, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 50017 to base position 60903.

The invention further provides a nucleotide sequence encoding a polyketide synthase comprising the functional domains KS, AT, DH, KR, ACP, KS, AT, DH, KR, ACP, KS, AT, KR, ACP, KS, AT, DH, KR, ACP, KS, AT, DH, KR, ACP, and responsible for biosynthesis of part of the polyketide moiety of the macrocyclic backbone. The amino acid sequence it codes for is as shown in SEQ ID NO: 11, designated as sfaH, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 60918 to base position 85823.

The invention further provides a nucleotide sequence encoding a polyketide synthase comprising the functional domains KS, AT, KR, ACP, KS, AT, KR, ACP, and responsible for biosynthesis of part of the polyketide moiety of the macrocyclic backbone. The amino acid sequence it codes for is as shown in SEQ ID NO: 12, designated as sfaI, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 85823 to base position 96040.

The invention further provides a nucleotide sequence encoding a zinc-finger dehydrogenase. The amino acid sequence it codes for is as shown in SEQ ID NO: 13, designated as sfaJ, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 96225 to base position 97391.

The invention further provides a nucleotide sequence encoding an iterative polyketide synthase comprising the functional domains KS, AT, ACP, KR. The amino acid sequence it codes for is as shown in SEQ ID NO: 14, designated as sfaK, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 97396 to base position 101943.

The invention further provides a nucleotide sequence encoding an acyltransferase. The amino acid sequence it codes for is as shown in SEQ ID NO: 15, designated as sfaL, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 101936 to base position 103213.

The invention further provides a nucleotide sequence encoding a short chain dehydrogenase/reductase. The amino acid sequence it codes for is as shown in SEQ ID NO: 16, designated as sfaM, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 103210 to base position 103929.

The invention further provides a nucleotide sequence encoding an acyl ketoacid synthase. The amino acid sequence it codes for is as shown in SEQ ID NO: 17, designated as sfaN, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 104001 to base position 105023.

The invention further provides a nucleotide sequence encoding a free acyl carrier protein. The amino acid sequence it codes for is as shown in SEQ ID NO: 18, designated as sfaO, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 105091 to base position 105345.

The invention further provides a nucleotide sequence encoding an asparagine synthase analogue. The amino acid sequence it codes for is as shown in SEQ ID NO: 19, designated as sfaP, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 105366 to base position 107216.

The invention further provides a nucleotide sequence encoding a free thioesterase. The amino acid sequence it codes for is as shown in SEQ ID NO: 20, designated as sfaQ, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 107366 to base position 108145.

The invention further provides a nucleotide sequence encoding a Crotonyl-coA reductase. The amino acid sequence it codes for is as shown in SEQ ID NO: 21, designated as sfaR, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 108150 to base position 109511.

The invention further provides a nucleotide sequence encoding a MbtH-family protein, the amino acid sequence it codes for is as shown in SEQ ID NO: 22, designated as sfaS, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 109583 to base position 109798.

The invention further provides a nucleotide sequence encoding a protein of unknown function. The amino acid sequence it codes for is as shown in SEQ ID NO: 23, designated as sfaV1, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 109776 to base position 110312.

The invention further provides a nucleotide sequence encoding another protein of unknown function. The amino acid sequence it codes for is as shown in SEQ ID NO: 24, designated as sfaV2, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 111285 to base position 111743.

The invention further provides a nucleotide sequence encoding another protein of unknown function. The amino acid sequence it codes for is as shown in SEQ ID NO: 25, designated as sfaV3, and the corresponding nucleotide sequence is as shown in SEQ ID NO: 1, starting from base position 112218 to position 112652.

The complementary sequence of SEQ ID NO: 1 may be obtained according to the principle of DNA base complementarity. The nucleotide sequence or part of the nucleotide sequence of SEQ ID NO: 1 may be obtained by polymerase chain reaction (PCR), or by suitable restriction enzyme digestion of corresponding DNA, or by other suitable techniques. The invention further provides a method to obtain a recombinant DNA plasmid comprising at least part of the DNA sequence in SEQ ID NO: 1.

The invention further provides a method to obtain a microorganism containing interrupted SFA biosynthetic genes, wherein at least one of the genes comprises the nucleotide sequence as shown in SEQ ID NO: 1.

The nucleotide sequence or part of the nucleotide sequence according to the invention may be obtained through a method based on polymerase chain reaction (PCR), or a gene similar to SFA biosynthetic gene can be obtained from other organisms by a Southern hybridization method or the like, using a DNA fragment comprising a sequence according to the invention as probe.

A cloned DNA comprising the nucleotide sequence or at least part of the nucleotide sequence according to the invention may be used to identify more library plasmids from the genomic library of Streptomyces sp. A92-308110 (S. flaveolus). These library plasmids comprise at least part of the sequence according to the invention, and comprise DNAs having not been cloned from the adjacent regions in the Streptomyces sp. A92-308110 (S. flaveolus) (S. flaveolus) genome as well.

Thus, for example, a nucleic acid or nucleotide sequence according to any aspect of the invention is a DNA.

The nucleotide sequence or at least part of the nucleotide sequence according to the invention may be modified or mutated. These methods includes insertion, replacement or deletion, polymerase chain reaction, error-prone polymerase chain reaction, site specific mutagenesis, re-ligation of different sequences, DNA shuffling with different parts of the sequence or with homologous sequences from other source, or mutagenesis by UV or chemical agent, etc.

The present invention also provides recombinant vectors such as DNA expression vectors comprising the above nucleic acids. Vectors typically comprise the aforementioned DNA together with one or more promoters or other regulatory elements. These vectors and methods of this invention enable one skilled in the art to generate recombinant host cells with the ability to produce polyketides. Thus the invention provides a method of preparing a polyketide, such as sanglifehrin A or a sanglifehrin A analogue, said method comprising culturing a transformed host cell, which host cell has been transformed with an expression vector comprising nucleic acids encoding all or part of the sanglifehrin gene cluster described in SEQ ID No. 1. A polyketide produceable by the aforemention method and which is not sanglifehrin A is also provided as an aspect of the invention. Desirably the vector comprises nucleic acid coding for a functional PKS which is able to generate sanglifehrin A or a sanglifehrin A analogue if cultured in an appropriate medium. In some embodiments the transformed host cell does naturally produce sanglifehrin A. In some embodiments the transformed host cell does not naturally produce sanglifehrin A. Such a transformed host cell is also provided as an aspect of the invention. A specific embodiment is a host cell transformed with a vector comprising nucleic acid encoding all or part of the sanglifehrin A biosynthesis gene cluster (e.g. it encodes the sanglifehrin A biosynthesis gene cluster exemplified by the nucleic acid of SEQ ID No. 1) which host cell does not naturally produce sanglifehrin A.

Thus, there are also provided:

-   -   A polypeptide or plurality of polypeptides encoded by the any of         the aforementioned nucleic acids;     -   A polyketide synthase encoded by the aforementioned nucleic acid         which encodes one or more polyketide biosynthesis proteins; and     -   A hybrid protein encoded by a modular nucleic acid or gene         cluster in which at least one domain or module or gene is not         native to the sanglifehrin A biosynthesis gene cluster.

In another embodiment, the invention provides an isolated polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos. 2-25; an isolated polypeptide consisting of at least 10, 50, 100, 200 or 500 consecutive amino acids of the polypeptides of SEQ ID Nos. 2-25; and an isolated polypeptide having at least 50%, 60%, 80%, 85%, 90%, 95%, 97% or 99% homology to the above sequences as determined by BLASTP (Altschul et al., 1990) with the default parameters.

The invention also provides a method of preparing a hybrid polyketide, said method comprising transforming a host cell with a recombinant vector comprising nucleic acids encoding all or part of the sanglifehrin gene cluster described in SEQ ID No. 1 in which at least one domain or module or gene is not native to the sanglifehrin A biosynthesis gene cluster, then culturing the transformed host cell.

The invention provides a polyketide produceable by the aforementioned process which is not sanglifehrin A.

The cloned gene comprising the nucleotide sequence or at least part of the nucleotide sequence according to the invention may by expressed in heterogeneous host by suitable expression system, to obtain corresponding or higher enzymatic or other biological activity or yield. These heterogeneous hosts include Streptomyces, Pseudomonas, E. coli, Bacillus, yeast, plants and animals, etc.

It is well known to those skilled in the art that polyketide gene clusters may be expressed in heterologous hosts (Pfeifer et al., 2001). Accordingly, the present invention includes the transfer of the sanglifehrin biosynthetic gene cluster with or without resistance and regulatory genes, either complete, engineered, containing mutations or containing deletions, for complementation in heterologous hosts. Methods and vectors for the transfer as defined above of such large pieces of DNA are well known in the art (Rawlings, 2001; Staunton and Weissman, 2001) or are provided herein in the methods disclosed.

In this context a preferred heterologous host cell strain is a prokaryote, more preferably an actinomycete or Escherichia coli, still more preferably include, but are not limited to S. hygroscopicus, S. hygroscopicus sp., S. hygroscopicus var. ascomyceticus, Streptomyces tsukubaensis, Streptomyces coelicolor, Streptomyces lividans, Saccharopolyspora erythraea, Streptomyces fradiae, Streptomyces avermitilis, Streptomyces cinnamonensis, Streptomyces rimosus, Streptomyces albus, Streptomyces griseofuscus, Streptomyces longisporoflavus, Streptomyces venezuelae, Micromonospora griseorubida, Amycolatopsis mediterranei or Actinoplanes sp. N902-109.

Thus, in a further aspect, the present invention provides novel strains wherein one or more of the genes coding for sanglifehrin biosynthesis are inactivated or deleted.

In a further aspect, the present invention provides novel strains where one or more modules or domains of the PKS genes sfaE, sfaF, sfaG, sfaH or sfaI are inactivated, deleted or replaced by non-native e.g. heterologous domains or modules. These strains may produce novel sanglifehrins. In particular there is provided an engineered strain based on an SFA producing strain wherein one or more PKS genes selected from sfaE, F, G, H and I have been modified whereby one or more domains or modules have been deleted, mutated so as to make inactive or less active an enzymatic function or so as have altered functionality or replaced by an replacement or whereby one or more non-native domains or modules have been inserted e.g. whereby one or more domains or modules from elsewhere in the SFA biosynthetis gene cluster or from a heterologous polyketide biosynthetic cluster have been inserted.

In a further aspect, the present invention provides novel strains where one or more modules or domains of the NRPS gene sfaD are inactivated, deleted or replaced by non-native e.g. heterologous domains or modules. These strains may produce novel sanglifehrins. In particular there is provided an engineered strain based on an SFA producing strain wherein NRPS gene sfaD has been modified whereby one or more domains or modules have been deleted, mutated so as to make inactive or less active an enzymatic function or so as have altered functionality or replaced by an replacement or whereby one or more non-native domains or modules have been inserted.

Further aspects of the invention include:

-   -   An engineered strain based on an SFA producing strain wherein         regulatory gene sfaC is modified so as to increase or decrease         its activity, or deleted, or a regulatory element associated         therewith (such as a promoter) that controls expression of sfaC         is modified, replaced or deleted. For example a strain wherein         regulatory gene sfaC or the control thereof is modified so as to         increase its activity or expression level may produce SFA (or an         SFA analogue) with greater yield. For example, sfaC may be         overexpressed in the strain by using a promoter for         overexpressing sfaC (such as permE) e.g. in a vector (such as         those described in Kieser et al, 1999) and optionally together         with a selectable marker (such as apramycin).     -   An engineered strain, not being a naturally SFA producing strain         (i.e. a heterologous host) containing the SFA biosynthetic gene         cluster under the control of one or more heterologous promoters.         By use of powerful promoters such a strain may be used to         produce SFA in high yields by means of a process involving         culturing the strain and optionally isolating SFA.     -   A method for producing higher levels of sanglifehrins involving         overexpressing sfaC.     -   An engineered strain based on an SFA producing strain wherein         one or more starter unit or precursor biosynthesis genes         selected from sfaA, B, J, M, N, P, Q, L and O have been deleted         or modified so as to decrease their activity or modified or         replaced so as to alter their substrate selectivity.

Aforementioned engineered strains based on an SFA producing strain may produce SFA or an SFA analogue if fed appropriately. Engineered strains may have any and all combinations of the above modifications and may have additional gene modifications.

The amino acid sequences according to the invention may be used to isolate a desired protein, and may be used for preparation of antibodies.

With certain amino acids deleted or substituted, polypeptides comprising the amino acid sequence or at least part of the sequence according to the invention may still have biological activity or even have new biological activity or desirable properties such as increased yield or optimized protein kinetics or others.

A gene or gene cluster comprising the nucleotide sequence or at least part of the nucleotide sequence according to the invention may be expressed in heterogeneous hosts, and their function in the host metabolic chain may be investigated by DNA Chip Technology.

A recombinant plasmid can be constructed by a genetic recombination from a gene or gene cluster comprising the nucleotide sequence or at least part of the nucleotide sequence according to the invention, to establish the biosynthetic pathway, or a method of insertion, replacement, deletion or inactivation can be used to establish the biosynthetic pathway.

A cloned gene or DNA fragment comprising nucleotide sequence or at least part of the nucleotide sequence according to the invention may be used to generate new structural analogues of SFA by interrupting one or several steps of SFA biosynthesis. The DNA fragment or gene may be used to increase the yield of SFA or derivatives thereof, the invention provides a method to increase yield in a genetically engineered microorganism. Examples of this include inactivation or deletion of negative regulators.

The non-ribosomal peptide synthetase according to the invention may be used to generate new peptide compounds, by deletion, insertion, alteration or inactivation of one or more domains, modules or genes of the non-ribosomal polypeptide synthetase from the same or a different non-ribosomal peptide synthetase system.

The polyketide synthase according to the invention may be used to generate new polyketides or hybrid polyketides, by deletion, insertion, alteration or inactivation of one or more domains, modules or genes of the polyketide synthase from the same or a different polyketide synthase system.

Fragments or genes comprising the nucleotide sequence or at least part of the nucleotide sequence according to the invention may be used to construct a library of nonribosomal peptide synthetases, or a library of derivatives of non-ribosomal polypeptide synthetases, or a combinatorial library.

Fragments or genes comprising the nucleotide sequence or at least part of the nucleotide sequence according to the invention may be used to construct a library of polyketide synthases, or a library of derivatives of polyketide synthases, or a combinatorial library.

In a word, all the gene and protein information associated with SFA biosynthesis provided herein can facilitate the understanding of biosynthesis mechanism of SFA natural products, and provides material and knowledge for further genetic engineering. The genes and their proteins provided herein may be used to search for and discover compounds, genes, or proteins that can be used in medicine, industry, or agriculture.

Additionally, in the present invention, specific changes have been made to the sanglifehrin biosynthetic gene cluster, with the original intention to understand the biosynthetic pathway, exemplify the inventions herein, and generate molecules for interrogation of the mechanism of immunosuppressant activity.

In general, sequence identity percentages may be determined by BLASTN (nucleic acid) or BLASTP (protein) (Altschul et al., 1990) with the default parameters.

Thus, in one aspect of the invention there is provided sanglifehrin analogue compounds of formula (I) or (II) or a pharmaceutically acceptable salt thereof:

wherein:

R₁ represents one of moieties A, B or C:

R₂ represents OH and R₃ represents H or R₂ and R₃ represent a bond;

R₄ represents OH and R₅ represents H or R₄ and R₅ represent a bond;

R₆ represents OH and R₇ represents H or R₆ and R₇ represent a bond;

R₈ represents OH and R₉ represents H or R₈ and R₉ represent a bond;

R₁₀ represents Me or CH₂CH₂C(O)CH₃;

R₁₁ represents H or Me;

with the proviso that when R₁₀ represents CH₂CH₂C(O)CH₃, then both R₂ and R₄ cannot represent OH and/or both R₆ and R₈ cannot represent OH,

and with the proviso that when R₁₀ represents CH₂CH₂C(O)CH₃, then R₂ and R₄, and R₆ and R₈ cannot all represent bonds.

In one embodiment, R₂ represents OH, R₃ represents H and R₄ and R₅, R₆ and R₇ and R₈ and R₉ represent bonds and R₁₀ represents CH₂CH₂C(O)CH₃

In one embodiment, R₅ represents OH, R₆ represents H and R₂ and R₃, R₆ and R₇ and R₈ and R₉ represent bonds and R₁₀ represents CH₂CH₂C(O)CH₃

In one embodiment, R₇ represents OH, R₈ represents H and R₄ and R₅, R₆ and R₂ and R₃ and R₉ represent bonds and R₁₀ represents CH₂CH₂C(O)CH₃

In one embodiment, R₈ represents OH, R₉ represents H and R₄ and R₅, R₆ and R₇ and R₂ and R₃ represent bonds and R₁₀ represents CH₂CH₂C(O)CH₃

Preferably R₁ represents moiety C.

Preferably R₁₁ represents H;

Specific embodiments include the following:

which may be expected to be produced by deletion/inactivation of sfaK optionally in conjunction with exchange of the AT of module 13 for an AT module which accepts methyl malonate in the SFA biosynthesis gene cluster;

which may be expected to be produced by deletion/inactivation of the DH of module 8 in the SFA biosynthesis gene cluster;

which may be expected to be produced by deletion/inactivation of the DH of module 10 in the SFA biosynthesis gene cluster;

which may be expected to be produced by deletion/inactivation of the DH of module 11 in the SFA biosynthesis gene cluster;

which may be expected to be produced by deletion/inactivation of the DH of module 7 in the SFA biosynthesis gene cluster;

which may be expected to be produced by deletion/inactivation of sfaK optionally in conjunction with exchange of the AT of module 13 for an AT module which accepts methyl malonate in the SFA biosynthesis gene cluster;

which may be expected to be produced by deletion/inactivation of the DH of module 8 in the SFA biosynthesis gene cluster;

which may be expected to be produced by deletion/inactivation of the DH of module 10 in the SFA biosynthesis gene cluster;

which may be expected to be produced by deletion/inactivation of the DH of module 11 in the SFA biosynthesis gene cluster;

which may be expected to be produced by deletion/inactivation of the DH of module 7 in the SFA biosynthesis gene cluster;

and pharmaceutically acceptable salts thereof.

The above structures show a representative tautomer and the invention embraces all tautomers of the compounds of formulas (I) and (II) for example keto compounds where enol compounds are illustrated and vice versa.

The invention embraces all stereoisomers of the compounds defined by formulas (I) and (II) as shown above.

In a further aspect, the present invention provides processes for production of sanglifehrin analogues such as those defined by formulas (I) or (II) above by culturing a sanglifehrin analogue producing strain and optionally isolating the compounds produced.

In a further aspect, the present invention provides sanglifehrin analogues such as compounds of formula (I) and (II) or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical.

The aforementioned compounds of formula (I) and (II) or a pharmaceutically acceptable salt thereof or a formulation thereof may be administered by any conventional method including topically (for example by inhalation, vaginally, intranasally, or by eye or ear drop), enterally (for example orally or rectally) or parenterally (for example by intravenous, intracavernosal, subcutaneous, intramuscular, intracardiac or intraperitoneal injection) or via a medical device (for example via a stent). The treatment may consist of a single dose or a plurality of doses over a period of time.

Whilst it is possible for sanglifehrin A or an analogue such as a compound of formula (I) and (II) or a pharmaceutically acceptable salt thereof to be administered alone, it is preferable to present it as a pharmaceutical composition, together with one or more pharmaceutically acceptable diluents or carriers. The diluents or carrier(s) must be “physiologically acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. In some cases, the diluent or carrier will be water or saline which will be sterile and pyrogen free.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the composition are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Compositions in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

Aerosol compositions suitable for administering via inhalation can also be made using methods known in the art. Examples of this include administration of the compounds of the invention by inhalation in the form of a powder (e.g. micronized) or in the form of atomized solutions or suspensions. The aerosol composition may be placed in a suitable pressurized propellant, and may be used with additional equipment such as nebulizer or inhaler.

For applications to external tissues, for example the mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active agent may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active agent may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

The compounds of the invention may also be administered using medical devices known in the art. For example, in one embodiment, a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. No. 5,399,163; U.S. Pat. No. 5,383,851; U.S. Pat. No. 5,312,335; U.S. Pat. No. 5,064,413; U.S. Pat. No. 4,941,880; U.S. Pat. No. 4,790,824; or U.S. Pat. No. 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

The compounds can be administered as the sole active agent, or in combination with other pharmaceutical agents.

In a further aspect, the present invention provides sanglifehrin analogues such as compounds of formula (I) and (II) or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of immune disorders, inflammatory disorders, cardiac diseases, viral diseases (such as HIV and HCV) and/or rejection of transplants.

In a further aspect, the present invention provides use of sanglifehrin analogues such as compounds of formula (I) and (II) or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of immune disorders, inflammatory disorders, cardiac diseases, viral diseases (such as HIV and HCV) and/or rejection of transplants.

In a further aspect, the present invention provides a method for the treatment or prevention of immune disorders, inflammatory disorders, cardiac diseases, viral diseases (such as HIV and HCV) and/or rejection of transplants which comprises administering to a subject (especially a human subject) a therapeutically effective amount of a sanglifehrin analogue such as a compound of formula (I) and (II) or a pharmaceutically acceptable salt thereof.

For completeness, we provide the following further aspects of the invention:

A biosynthetic gene cluster of an immunosuppressant sanglifehrin A, characterized in that it comprises 24 genes responsible for sanglifehrin A biosynthesis, which are:

-   1) one non-ribosomal peptide backbone synthetase (NRPS) gene, sfaD,     wherein: -   sfaD locates in the nucleotide sequence of the gene cluster at bases     19885-30714, is 10830 base pairs in length, and encodes a     non-ribosomal peptide synthetase of 3609 amino acids; -   2) five type-I linear polyketide synthase (PKS) genes, sfaE, sfaF,     sfaG, sfaH, sfaI, wherein: -   sfaE locates in the nucleotide sequence of the gene cluster at bases     30707-37360, is 6654 base pairs in length, and encodes a polyketide     synthase of 2217 amino acids; -   sfaF locates in the nucleotide sequence of the gene cluster at bases     37394-50014, is 12621 base pairs in length, and encodes a polyketide     synthase of 4206 amino acids; -   sfaG locates in the nucleotide sequence of the gene cluster at bases     50017-60903, is 10887 base pairs in length, and encodes a polyketide     synthase of 3628 amino acids; -   sfaH locates in the nucleotide sequence of the gene cluster at bases     60918-85823, is 24906 base pairs in length, and encodes a polyketide     synthase of 8301 amino acids; -   sfaI locates in the nucleotide sequence of the gene cluster at bases     85823-96040, is 10218 base pairs in length, and encodes a polyketide     synthase of 3405 amino acids; -   3) one type-I iterative polyketide synthase gene, sfaK, wherein: -   sfaK locates in the nucleotide sequence of the gene cluster at bases     97396-101943, is 4548 base pairs in length, and encodes a polyketide     synthase of 1515 amino acids; -   4) ten functional genes for precursor synthesis, sfaA, sfaB, sfaJ,     sfaM, sfaN, sfaP, sfaQ, sfaR, sfaL, sfaO, wherein: -   sfaA locates in the nucleotide sequence of the gene cluster at bases     17024-17854, is 831 base pairs in length, and encodes a     phenylalanine meta-hydroxylase of 276 amino acids; -   sfaB locates in the nucleotide sequence of the gene cluster at bases     17851-19191, is 1341 base pairs in length, and encodes a     N5-ornithine oxygenase of 446 amino acids; -   sfaJ locates in the nucleotide sequence of the gene cluster at bases     96225-97391, is 1167 base pairs in length, and encodes a zinc-finger     dehydrogenase of 388 amino acids; -   sfaM locates in the nucleotide sequence of the gene cluster at bases     103210-103929, is 720 base pairs in length, and encodes a short     chain dehydratase/reductase of 239 amino acids; -   sfaN locates in the nucleotide sequence of the gene cluster at bases     104001-105023, is 1023 base pairs in length, and encodes a     ketosynthase of 340 amino acids; -   sfaP locates in the nucleotide sequence of the gene cluster at bases     105366-107216, is 1851 base pairs in length, and encodes an     asparagine synthase analogue of 616 amino acids; -   sfaQ locates in the nucleotide sequence of the gene cluster at bases     107366-108145, is 780 base pairs in length, and encodes a     thioesterase of 259 amino acids; -   sfaR locates in the nucleotide sequence of the gene cluster at bases     108150-109511, is 1362 base pairs in length, and encodes a     crotonyl-coA reductase of 453 amino acids; -   sfaL locates in the nucleotide sequence of the gene cluster at bases     101936-103213, is 1278 base pairs in length, and encodes an     acyltransferase of 425 amino acids highly homologous to the     transacylation domain in polyketide synthase; -   sfaO locates in the nucleotide sequence of the gene cluster at bases     105091-105345, is 255 base pairs in length, and encodes an acyl     carrier protein of 84 amino acids; -   5) two regulatory genes, sfaC and sfaS, wherein: -   sfaC locates in the nucleotide sequence of the gene cluster at bases     19193-19888, is 696 base pairs in length, and encodes a     transcription regulatory factor of 231 amino acids; -   sfaS locates in the nucleotide sequence of the gene cluster at bases     109583-109798, is 216 base pairs in length, and encodes a MbtH     factor of 71 amino acids; -   6) five genes encoding functionally unknown proteins, sfaU1, sfaU2,     sfaV1, sfaV2, sfaV3, wherein: -   sfaU1 locates in the nucleotide sequence of the gene cluster at     bases 14973-15413, is 441 base pairs in length, and encodes a     functionally unknown protein of 146 amino acids; -   sfaU2 locates in the nucleotide sequence of the gene cluster at     bases 15596-16063, is 468 base pairs in length, and encodes a     functionally unknown protein of 155 amino acids; -   sfaV1 locates in the nucleotide sequence of the gene cluster at     bases 109776-110312, is 537 base pairs in length, and encodes a     functionally unknown protein of 178 amino acids; -   sfaV2 locates in the nucleotide sequence of the gene cluster at     bases 111285-111743, is 459 base pairs in length, and encodes a     functionally unknown protein of 152 amino acids; -   sfaV3 locates in the nucleotide sequence of the gene cluster at     bases 112218-112652, is 435 base pairs in length, and encodes a     functionally unknown protein of 144 amino acids.

The non-ribosomal peptide synthetase in the sanglifehrin A biosynthetic gene cluster, characterized in comprising the following modules or domains: peptidyl condensing-enzyme domain C, adenylation domain A, peptidyl carrier protein PCP, and a termination domain C for terminal group cyclization.

The polyketide synthase in the sanglifehrin A biosynthetic gene cluster, characterized in comprising the following modules or domains: keto condensing domain KS, transacylation domain AT, acyl carrier protein domain ACP, dehydration domain DH, keto reduction domain KR, and enol reduction domain ER.

A use of any protein encoded by the sanglifehrin A biosynthetic gene cluster, for the catalytic synthesis of the immunosuppressant sanglifehrin A and any corresponding analogues of its family.

A use of any protein encoded by the sanglifehrin A biosynthetic gene cluster, for the catalytic synthesis of the backbone of a hybrid polyketide-nonribosomal peptide.

The use of the sanglifehrin A biosynthetic gene cluster, wherein a genetic modification is made, and the biological fermentation of the mutant obtained produces non-natural analogues of sanglifehrin A, such as those hydroxylated at position 21 and 25.

The use of the sanglifehrin A biosynthetic gene cluster, wherein a genetic modification is made, and the biological fermentation of the mutant obtained produces compensatory products, non-natural analogues of sanglifehrin A which are substituted with methyl group at position 14.

DESCRIPTION OF THE FIGURES

FIG. 1: Chemical structures of sanglifehrins (sanglifehrin A and analogues B, C and D).

FIG. 2: Gene organization and restriction map of SFA biosynthetic gene cluster. (A) four overlapped cosmids representing the ˜150 kb DNA region of the Streptomyces sp. A92-308110 (S. flaveolus) genome, B represents restriction enzyme BamHI, the solid line represents the parts that have already been DNA sequenced, Probe-P1 to P4 represent labeled probes; (B) the genetic organization of SFA biosynthetic gene cluster. Unknown: unknown gene; PKS: polyketide synthase gene; NRPS: non-ribosomal peptide synthetase gene; Beyond Cluster: gene outside boundary; Transposase: transposon; Modification: precursor synthesis gene

FIG. 3: Proposed biosynthetic pathways for each building block of SFA. (A) piperazic acid; (B) starter unit; (C) special extension unit. In steps marked with SfaX, X corresponds to the protein encoded by the respective gene in gene cluster as described herein; Marking a step with symbol “?” means that it is not yet clear whether the step is performed by an enzyme encoded by a gene of this gene cluster, or is performed in vivo by help of an enzyme involved in primary metabolism; A step without any mark means that it may be performed by help of a primary metabolism process.

FIG. 4: Pathway for SFA backbone formation. PKS: polyketide synthase; NRPS: non-ribosomal peptide synthetase; KS: functional domain for keto synthesis; AT: functional domain for acyl transfer; KR: functional domain for keto reduction; ER: functional domain for enol reduction; DH: functional domain for dehydration; ACP: acyl carrier protein; C: functional domain for peptide condensation; A: functional domain for adenylation; PCP: peptidyl carrier protein.

FIG. 5: High-performance liquid chromatography (HPLC) analysis of fermentation products derived from the interruption of the sequence of SFA biosynthetic gene cluster by cloned probe. WT: wild type; Standard: SFA standard; Mutant: mutant strain.

FIG. 6: High-performance liquid chromatography (HPLC) analysis of fermentation products of a mutant strain resulted from gene substitution. (A) wild type, (B) knock-out mutant of zinc binding dehydrogenase gene sfaJ, (C) knock-out mutant of iterative linear polyketide synthase gene sfaK, (D) knockout mutant of acyltransferase gene sfaL, (E) knockout mutant of short chain dehydrogenase gene sfaM, (F) knockout mutant of phenylalanine hydroxylase gene sfaA, (G) knockout mutant of ornithine oxygenase gene sfaB, (H) knockout mutant of fatty acid ketosynthase gene sfaN, (I) knockout mutant of asparagine synthase analogue gene sfaP, (J) knockout mutant of Crotonyl-coA reductase gene sfaR, (K) knockout mutant of left flanking unknown protein sfaU1, (L) knockout mutant of right flanking TetR regulatory factor, (M) DH domain of module 8 site-directed mutagenesis, (N) DH domain of module 10 site-directed mutagenesis. Streptomyces sp. A92-308110 (S. flaveolus); mv.: mutant strain (a mark used to distinguish from wild type); tsekangensis: T mutant (produces SFT); hasangensis: H mutant (produces SFH); xuwengensis: X mutant (produces SFX)

FIG. 7: Summary figure containing proposed biosynthetic pathway

The module numbering of FIG. 3 has been superceded by that of FIG. 7. Therefore when module numbers are referred herein the module numbers are those referred to in FIG. 7.

DESCRIPTION OF THE SEQUENCES OF THE SEQUENCE LISTING

SEQ ID No. 1: nucleic acid sequence of the SFA biosynthetic gene cluster

SEQ ID No. 2: amino acid sequence of SfaU1

SEQ ID No. 3: amino acid sequence of SfaU2

SEQ ID No. 4: amino acid sequence of SfaA

SEQ ID No. 5: amino acid sequence of SfaB

SEQ ID No. 6: amino acid sequence of SfaC

SEQ ID No. 7: amino acid sequence of SfaD

SEQ ID No. 8: amino acid sequence of SfaE

SEQ ID No. 9: amino acid sequence of SfaF

SEQ ID No. 10: amino acid sequence of SfaG

SEQ ID No. 11: amino acid sequence of SfaH

SEQ ID No. 12: amino acid sequence of SfaI

SEQ ID No. 13: amino acid sequence of SfaJ

SEQ ID No. 14: amino acid sequence of SfaK

SEQ ID No. 15: amino acid sequence of SfaL

SEQ ID No. 16: amino acid sequence of SfaM

SEQ ID No. 17: amino acid sequence of SfaN

SEQ ID No. 18: amino acid sequence of SfaO

SEQ ID No. 19: amino acid sequence of SfaP

SEQ ID No. 20: amino acid sequence of SfaQ

SEQ ID No. 21: amino acid sequence of SfaR

SEQ ID No. 22: amino acid sequence of SfaS

SEQ ID No. 23: amino acid sequence of SfaV1

SEQ ID No. 24: amino acid sequence of SfaV2

SEQ ID No. 25: amino acid sequence of SfaV3

SEQ ID Nos. 26-29: Primer sequences described in Example 1

SEQ ID Nos. 30 and 31: Primer sequences described in Example 11

SEQ ID Nos. 32-47: Primer sequences described in Example 12

SEQ ID Nos. 48-63: Primer sequences described in Example 14

SEQ ID Nos. 64-67: Primer sequences described in Example 16

General Methods

In general, methods were used as described in Maniatis et al., 1998, Sambrook and Russell, 2001 or in Kieser et al., 1999. Examples or alternative methods are detailed below.

Bacterial Strains and Plasmids

Escherichia coli DH10B (GibcoBRL) was grown in 2×TY medium as described by Sambrook and Russell (2001) and E. coli ET12567 (pUZ8002) as described in Paget et al. (1999) in 2×TY medium with kanamycin (25 mg/L) and chloramphenicol (12.5 mg/L). E. coli transformants were selected for with 100 mg/L ampicillin or 50 mg/L apramycin.

Most plasmids used are described in Kieser et al., 1999 and Sambrook et al., 2001. pTV1, also known as pBS3030, is described in Cheng et al., 2003, pIJ773 is described in GenBank accession no. AX657066.1. pANT841 is described in Genbank: AF438749

The sanglifehrin producer Streptomyces sp. A92-308110 (S. flaveolus) may be obtained from the DSMZ, Braunschweig, Germany as Streptomyces sp. DSM 9954.

Media Recipes

AS-1 Agar Medium:

g/100 mL Yeast extract 0.1 L-Alanine 0.02 L-Arginine 0.05 Soluble starch 0.5 NaCl 0.25 Na₂SO₄ 1.0 Agar 2.0

Distilled water to 100 ml

Medium is adjusted by NaOH to pH 7.5;

ISP4 Agar Medium:

g/L Soluble starch 10 K₂HPO₄ 1 MgSO₄•7H₂O 1 NaCl 1 (NH4)₂SO₄ 2 CaCO₃ 2 ISP trace salts solution 1 mL Agar 20

Distilled water to 1000 ml, adjust to pH 7.2 prior to sterilisation

ISP Trace Salt Solution:

g/L FeSO₄7H₂O 1 MnCl₂•4H₂O 1 ZnSO₄•7H₂O 1

Distilled water to 1000 ml

IWL-4 Agar Medium:

IWL-4 agar medium was prepared by adding to each liter of ISP-4 medium 1 g tryptone and 0.5 g yeast extract, and adjusting the medium to pH 7.2 by NaOH

Conjugation General Method

Donor bacteria, E. coli S17-1 containing the plasmid of interest was inoculated into test tubes in 3-4 mL LB broth under supplemented with 50 μg/mL apramycin with shaking overnight. 500 μL was then inoculated into 50 mL LB supplemented with 50 μg/ml apramycin in 250 mL flasks at 37° C. and grown to an OD600 of 0.5. The cells were recovered in a 50 mL EP tube by centrifugation at 3800 rpm for 10 min. They were then re-suspended with 20 mL LB by vortexing and recovered again. This was repeated two times and the recovered cells re-suspended in 1 mL LB. Acceptor bacteria Streptomyces sp A92-309110 (S. flaveolus) were centrifuged at 12000 rpm 3 min to recover the spores and re-suspended with in 500 μL of 0.1 M TES twice, then heat shocked at 50° C. for 10 min and 500 μL TSB broth added. This was then The spores were then incubated at 37° C. for 4-5 h. The spores were recovered and resuspended in 1 mL LB. The bacteria were mixed in ratios from 1:99 to 99:1, with 1:1 generally yielding a successful transformation. The mixed bacteria were spread on MS agar and the plates dried in a laminar flow cabinet. The plates were then incubated at 30° C. for 14-16 h. The plates were spread again with 3-4 mL sterile water to remove most of the E. coli and the plates dried for 1 h in a laminar flow cabinet. The plates were then overlaid with 1 mL sterile water containing apramycin to at 1 mg/mL and nalidixic acid to at 1 mg/mL and incubated at 30° C. for 3-5 days. One or two or more single clones were selected and inoculated into 3 mL TSB broth with apramycin at 30 μg/mL with shaking at 30° C. for several days. 100 μL of fresh culture were spread onto ISP-4 agar and a double crossover allowed to occur by incubation for 2-3 days.

Total DNA Extraction from Streptomyces Sp A92-309110 (S. Flaveolus)

Streptomyces sp A92-309110 (S. flaveolus) spore suspensions stored at −80° C. were inoculated into 3 mL YEME medium. The culture was incubated for 12 h at 30° C., transferred into 1.5 mL EP tubes in two portions, centrifuged, and bacteria were recovered. Bacteria were resuspended in 500 μL STE solution; lysozyme was added to a final concentration of 4 mg/mL, and then incubated in a water bath at 37° C. for 30-45 min. After the bacteria had turned to are observed to become transparent and gel-like substance. Then, 250 μL of 2% SDS and 60 μL of 5 mol/L KAc were added, and well-mixed, frozen at −20° C. for 10 min, then centrifuged at 12000 rpm for 10 min. Supernatants were transferred into new EP 1.5 mL tubes. 500 μL 1:1 (v/v) phenol/chloroform mixture were added, and centrifuged at 10000 rpm for 3 min. This step was repeated several times until no more white denatured protein appeared at the interface. Then chloroform alone was used for another a final extraction. The supernatant was removed, well-mixed with an equal volume of isopropanol, left at room temperature for 30 min ro precipitate DNA, and centrifuged at 10000 rpm for 5 min. After washing with 70% alcohol and drying by suction filter, 200 μL TE buffer and 10 μL 10 mg/mL DNase-free RNase were added, and frozen at −20° C. for storage.

Fermentation of the SFA Producing Strain Streptomyces Sp A92-309110 (S. Flaveolus), and Related Strains, Product Isolation, Purification, and Identification

a) Liquid Fermentation

From a spore stock 50 μl (average concentration 1×10⁸ per ml) was transferred into 3 mL TSB (Tryptic Soytone Broth, Sigma) under appropriate antibiotic pressure (apramycin 50 μg/ml or erythromycin 50 μg/ml depending on the resistant marker of the mutant) and was shaken (at 27° C., 250 rpm) for 30 h. From the primary culture 1 ml was transferred into 50 ml seed medium in 250 ml Erlenmeyer flask (diameter, 8.5 cm at bottom and 3.5 cm at mouth; height, 15 cm) and shaken (at 27° C., 250 rpm).

From the seed culture 5 mL was transferred into 90 ml production medium in 500 ml Erlenmeyer flask (diameter: 10.5 cm at bottom and 4 cm at mouth, height: 18 cm) and grown for 24 h at 25° C. (250 rpm), at which time 10 ml fresh production medium containing 4 g resin XAD-16 was added to the production culture (total 4 g XAD-16 in 100 mL production medium). The culture was incubated at the above conditions (25° C., 220 rpm), for further 3 days.

After fermentation was completed on day 4, the fermentation broth was passed through a paper filter aided by suction. The paper filter with the material remaining on it was transferred to a beaker, and frozen at −80° C. for 30 min, and freeze-dried overnight. 80-100 mL methanol was added to the dried material, stirred for 40 min, and filtered using suction. The filtered material was extracted with methanol for the second time, and filtered using suction. The resin was discarded, and the methanolic solutions were pooled, and dried under vacuum at 35-37° C. After evaporation to dryness, the material was transferred using two aliquots of 750 μL methanol. The two methanolic solutions were pooled and 1.5 mL aliquots were transferred to Ependurf tubes, and frozen at −20° C. for 1 h. The tubes were centrifuged at 12000 rpm, 4° C., for 10 min. The supernatant was transferred to new Eppendorf tubes, dried under vacuum until approximately 500 μL of liquid remained, which was then stored at 4° C.

Approximately 300 μL of the crude extract obtained as explained above was purified through flash column chromatography (RP-18), which was pre-rinsed with 40 mL of 40% acetonitrile in water. The column was then eluted with 40 mL of 50% acetonitrile in water, and 40-mL fractions were collected in separate tubes, and then concentrated. Pre-treated samples were further purified using LC-MS (using 80% acetonitrile in water containing 1% formic acid) for isolation and identification.

b) Solid Fermentation

For inoculation of several agar plates, 40 μL of spore stock was transferred to 3 mL TSB medium, and incubated overnight at 37° C. (while shaking at 250 rpm). From this overnight culture, 400-500 μL was transferred and spread evenly onto the surface of agar plates (150 mm) made either from R2YE, IWL-4, ISP-4, or AS-1 agar medium. Alternatively, aliquots (50 μL) of spore stock can be used to directly inoculate the agar plates. The inoculated agar plates were incubated at 30° C. for 6-7 days.

At the end of the incubation period, the culture on the agar medium was harvested into a 250 mL beaker and minced. 200 mL methanol was added to the material from one agar plate and the mixture was stirred for 2 h, then filtered through filter paper aided by suction. Solid materials were discarded, and the filtrate was dried in a rotary evaporator. In order to recover the fermentation product, two 500 μL aliquots of methanol were used to resuspend and transfer the dried material to 1.5 mL Eppendorf tubes. Purification of material from the methanolic concentrate followed a similar procedure as explained above for the liquid fermentation process. However, because there the content of water in solid media is low the freeze-drying step can be omitted. Samples were further purified using LC-MS (using 80% acetonitrile in water containing 1% formic acid) for isolation and identification.

Cloning SFA Biosynthetic Genes by PCR

The PCR system comprises: DMSO (8%, v/v), MgCl₂ (25 mM), dNTP (2.5 mM), degenerate primers (10 μM), Taq DNA polymerase (2.5 u), and a suitable amount of the total DNA of Streptomyces sp A92-309110 (S. flaveolus) as a template. First, 95° C., 3 min, 1 cycle; then, 94° C., 1 min, 68° C., 1 min, 72° C., 2 min, 5 cycles; 94° C., 1 min, 65° C., 1 min, 72° C., 2 min, 30 cycles; finally, 72° C., 10 min, 1 cycle. After PCR was finished, 1% Agarose Gel Electrophoresis was use to examine the results. DNA fragments of desired sizes were recovered from low melting point gel, and ligated with pGEM T Easy vector. E. coli DH5α competent cells were transformed, and spread on LB plate containing ampicillin, IPTG (Isopropyl-β-D-thiogalactopyranoside), and X-gal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside) were used for blue-white selection. White bacterial colonies were picked for overnight culture, and plasmids were extracted. It was assessed by EcoRI digestion whether plasmids contained inserted DNA fragments of desired sizes. These plasmids were then sequenced.

Nucleic Acid Hybridization

Several microliters of mycelium or spores of the mutant strain to be assessed were inoculated into 3 mL TSB medium, shaken at 220 rpm, and cultured at 30° C. until the culture medium thickened. Then genomic DNA was extracted. Based on sequence analysis, a hybridization strategy was designed, probes were prepared, and suitable restriction enzyme sites were selected for genomic DNA digestion.

15 μL of probe (containing 0.5-3 μg DNA) was incubated in a boiling water bath for 10 min, then transferred immediately into a salt-ice bath to cool. 2 μL Hexanucleotide Mix (10×), 2 μL dNTP Labeled Mix, 1 μL Klenow Enzyme Labeled were sequentially added and well-mixed, then the mixture was incubated in a water bath at 37° C. for 16 h. 0.8 μL 0.8M EDTA (pH=8.0) was added to stop the reaction, and 2.5 μL 4M LiCl was added and well-mixed. 75 μL pre-chilled anhydrous alcohol was added for precipitation. The mixture was frozen at −80° C. for 40 min, and centrifuged at 12000 rpm, 4° C. for 20 min. DNA was collected, washed with 70% prechilled alcohol, vacuum dried and dissolved in 50 μL TE buffer and stored at 4° C. After electrophoresis, the agarose gel was soaked in 0.25M HCl for 20 min, rinsed with deionized water, and then soaked for another 20 min with gentle shaking after the basic buffer was added. After exchanging the buffer once, the gel was soaked for another 20 min. After washing several times with deionized water, DNA was transferred to a nylon membrane. Transfer apparatus (BioRad) was used for the transfer. Following the instructions of the apparatus, one piece of wet filter paper was placed on the bottom plate, then overlaid with a nylon membrane of suitable size. The membrane should be 1 cm larger than the gel on each side, then a plastic membrane was overlaid and the clamps are fixed. The agarose gel was treated as described above and then placed on the membrane pore. After the apparatus is connected with the vacuum pump, the pressure is adjusted to maintain at 5-8 mmHg. After making a vacuum-tight seal, around 1 L 10×SSC hybridization buffer is added, and the liquid surface should cover the agarose gel. The transfer is performed for 2 h. Fixing may then be performed by baking for 40 min in oven at 120° C., or alternatively by exposing to UV light (2 J/cm2). The membrane is then stored in a dark and cool place before use. The fixed nylon membrane was placed into hybridization tubes, and hybridization solution was added. Pre-hybridization was performed at 64° C. for 30 min, then probes were added, and hybridization was performed at the desired temperature for 16 h. The membrane was sequentially washed twice for 5 min with 2×SSC stringent washing buffer at room temperature; then washed twice for 15 min with 0.5×SSC stringent washing buffer at 64° C. After stringent washing, the nylon membrane was first equilibrated for 1-5 min with washing buffer, and then incubated with blocking solution for 30 min, then incubated with antibody solution for 30 min. After 1-2 washings with washing buffer, it was equilibrated for 2-5 min with detection buffer. Developing buffer was added, and the membrane was allowed to develop in the dark. When a suitable intensity was reached, the membrane was washed with deionized water to stop the reaction, and hybridization was completed.

EXAMPLES

Further description will be made to the invention with reference to the accompanying figures in the following context.

Example 1 Cloning the Crotonyl-CoA Reductase Gene Fragment from the SFA Gene Cluster

Hitherto, whilst the total synthesis of SFA has been successfully completed, its natural production pathway is barely known, particularly with respect to the mechanism of formation of the N—N bond of the piperazic acid unit, and the unusual polyketide extension unit that probably utilises an iterative polyketide synthase. For the past few years, isotope-labeled studies have shown that glutamic acid and glutamine may be the biosynthetic precursors of piperazic acid in certain systems that produce natural products comprising piperazic acid units (Umezawa et al., 2001; Miller et al., 2007). According to analysis of existing gene clusters and of other reported gene clusters for natural products comprising piperazic acid, the possibility of ornithine being a substrate is not excluded; moreover, the unusual extension unit, as a side substituent on the macrocyclic backbone, may be formed based on an unusual iterative polyketide synthase. The above-mentioned two units may play important roles in developing new drugs and studying structure-activity relationships. Thus, there is a need for analysis of the SFA biosynthetic gene cluster to elucidate the biosynthesis mechanism of SFA, and then to develop its potential pharmaceutical value.

Based on analysis of the hybrid PKS-NRPS structure for the macrocyclic backbone, the gene cluster was assumed to possess a long linear PKS-NRPS region, with no known homologues, leading to an increase in the difficulty of isolation and cloning of the biosynthetic gene cluster. Indeed, by probing with elements of PKS and NRPS genes, DNA from 11 PKS and 7 NRPS clusters was isolated, then used for gene inactivation studies. All of these led to no effect on sanglifehrin production. Eventually, the inventors managed to clone the gene cluster using other specific units as probes. By analysis of naturally occurring biosynthesis gene clusters for polyketide natural products which comprise ethyl group-based structures, the inventors noted that ethyl units were generally introduced by using ethylmalonyl-CoA as a building block. The inventors conceived that the SFA gene cluster may contain a CCR homologue. Therefore, degenerate CCR primers were designed to clone the sequence of the highly conserved region of the crotonyl-coA synthetase responsible for biosynthesis of ethyl unit, which we thought may be present in S. sp. A92-308110 (S. flaveolus) genome. The cloned sequence was then labeled as a probe to perform library screening. The sequences of degenerate primers were as follows:

CCR Long-For (SEQ ID: 26): AGGAAT TCATGG CCTCCK CSRTSA ACTACA AY, CCR Long-Rev (SEQ ID: 27): TCGGAT CCGCCG AAGTTS GTCTCR WABCCC CA; CCR Short-For (SEQ ID: 28): AGGAAT TCGACA TCGACA TSGTBW TCGAG CA, CCR Short-Rev (SEQ ID: 29) TCGGAT CCGATG ATGCGC TTSWSB KDCATC CA.

Using these primers, two sequences of 900 bp and 300 bp were amplified from the Streptomyces sp A92-309110 (S. flaveolus) genome. The gel was cut, and the PCR fragments were recovered and digested by EcoRI and BamHI, and then cloned into pSP72 at EcoRI/BamHI site. The recombinant plasmids were identified by enzymatic digestion and sequencing. In total, three different sets of CCR gene fragments which were highly homologous to each other were cloned.

Example 2 Cloning, Sequence Analysis and Function Analysis of SFA Biosynthetic Gene Cluster

Fragments cloned as described above in example 1 were labeled with digoxin, and library screening was performed. The resulting 3 sets of cosmids obtained were re-divided into groups, and their relative positions were established according to restriction mapping. The 6.4 kb BamHI end fragment of pTL3101 (cQXD03-126-6) was chosen as probe to perform chromosome walking, and cosmid pSL36 was obtained. Then 1.4 kb BamHI fragment was chosen to perform chromosome walking, and cosmids pTL3104 (cQXD04-49-1˜50) were obtained. Then the BamHI fragment at the far left side was chosen to perform walking and cosmids pTL3106 (cQXD04-64-1˜40) were obtained. After these three rounds of chromosome walking, a total of about 150 kb of chromosome DNA was cloned. Using PKS and NRPS degenerate primers respectively, PCR assays were performed on some cosmids, from some of which specific bands were amplified. This confirmed that the gene cluster comprises CCR and hybrid PKS-NRPS.

TABLE 1 Sequence-based function analysis of the SFA biosynthetic gene cluster function of amino homologous similarity/ gene acid similar protein protein identity sfaU1 150 ZP_03193264 unknown protein 60/45 sfaU2 112 AAW49302 resolvase/integrase 81/75 sfaA 276 YP_001509705 Phenylalanine 70/57 meta-hydroxylase sfaB 446 YP_001852050 ornithine 73/61 N5-oxygenase sfaC 231 YP_001852059 transcription 73/60 regulatory factor sfaD 3609 YP_631823 non-ribosomal peptide 57/43 synthetase sfaE 2217 ABC87510 polyketide synthase 65/52 sfaF 4206 ABB88533 polyketide synthase 59/48 sfaG 3628 NP_824075 polyketide synthase 63/54 sfaH 8301 ZP_03174068 polyketide synthase 63/52 sfaI 3405 DQ_450945 polyketide synthase 73/65 sfaJ 332 YP_112153 zinc-binding 67/51 dehydrogenase sfaK 1515 YP_001104567 iterative linear 58/47 polyketide synthase sfaL 425 YP_480040 acyltransferase 54/42 sfaM 239 YP_949278 short chain 60/43 dehydrogenase sfaN 340 YP_001537010 ketosynthase 66/52 sfaO 84 AAD_20269 acyl carrier protein 61/45 sfaP 616 BAB12569 asparagine synthase 77/68 analogue sfaQ 259 CAQ64680 thioesterase 70/58 sfaR 453 YP_002204695 Crotonyl-coA 91/86 reductase sfaS 71 YP_882427 MbtH protein 82/67 sfaV1 178 CAD18995 unknown protein 81/65 sfaV2 152 YP_002199237 hypothetical protein 63/53 sfaV3 144 YP_001159721 hypothetical protein 57/45

The three cosmids with the highest coverage were selected to undergo full-length sequencing, and the inventors obtained 118,372 bp of continuous nucleotide sequence. Bioinformatics analysis revealed that it comprised 44 open reading frames (ORFs), at least 19 ORFs of which were suggested to be related to SFA biosynthesis, including: 1 NRPS gene (sfaD) responsible for synthesis of macrocyclic tripeptide backbone; 5 PKS genes (sfaE-I) which complete synthesis of the macrocyclic polyketide backbone; 11 putative precursor synthesis genes (sfaA-B, sfaJ-R), responsible for synthesis of the starter unit and each condensation unit, and for backbone modification; 2 regulatory genes (sfaC and sfaS) involved in regulation of SFA biosynthesis. Results of function analysis of each gene were shown in the table above.

Example 3 Determination of the Boundaries of the SFA Biosynthetic Gene Cluster

According to sequence analysis, at about 40 kb from the upstream sequencing start point, there is an incomplete trace of transposition (upstream of sfaU1 and sfaU2); at about 20 kb from the downstream sequencing end point, there are obviously two transposase genes (i.e. sfaT2 and sfaT3), but part of the upstream transposition gene is missing. Using the two sequences described above as boundaries, all the ORFs may be divided into three sections:

The about 120 kb sequence in the middle is the core responsible for SFA backbone biosynthesis, as described above.

The upstream section comprises 16 genes that are putatively not relevant with SFA biosynthesis, including 9 functional genes (cytochrome p450 mono-oxygenase gene orf16, acyl carrier protein gene orf14, adenylase gene orf13, methyltransferases gene orf12, cytochrome p450 hydroxylase gene orf11, deoxy guanine kinase gene orf10, dehydrogenase gene orf9 and proteinase M23S gene orf6), 5 genes encoding unknown proteins (orf15, orf7, orf5, sfaJ1, and sfaJ2), 2 genes encoding regulatory factors (orf4 and orf8, which belong to TetR and LysR family respectively), and 1 glycosyl transfer coupled protein gene (orf3).

The downstream section comprises genes that are putatively not relevant with SFA biosynthesis, including 3 genes encoding unknown proteins (sfaV1, sfaV2, and sfaV3) and 2 transposase genes (sfaT2 and sfaT3). There is also 1 gene encoding TetR family regulatory factor (orf23), 1 gene encoding TetR-coupled membrane protein (orf24), 1 FAD mono-oxygenase gene (orf25), and 1 incomplete endonuclease gene (orf26).

Preliminary analysis of the gene cluster sequence helped the inventors to determine the putative boundaries of the SFA biosynthetic gene cluster. The left boundary is located just upstream of sfaA, including 2 functionally unknown genes (sfaU1 and sfaU2). The right boundary is located just downstream of the MbtH gene, including 3 functionally unknown genes and 2 transposase genes (sfaV1˜3, orf21 and 22). As MbtH family proteins are often linked with NRPSs, the inventors presume that it is involved in the regulation of precursor biosynthesis. However, the activity of proteins of this family can be complemented by homologues elsewhere in the cluster; that is to say, all of the MbtH genes present in the whole genome would need to be knocked out, otherwise it will be complemented by its counterpart located at other site in the genome. As the total amount of MbtH in host bacteria was unknown, the downstream unknown protein and transposon were chosen for analysis. In addition, at 82 bp from the start of orf10, there is a 35 bp promoter sequence; and at 52 bp from where orf18 ends, there is a part of a ρ-independent terminator sequence. The whole gene cluster is located in the large transcriptional unit as described above. Based on the following analysis of the downstream transposases, it is presumed that this gene cluster comes from horizontal gene transfer.

3.1 Study of the Left Boundary

Study of the left boundary was focused on sfaU1 and sfaU2. sfaU1 has a full length of 453 bp, it encodes a functionally unknown protein of 150 amino acid residues, and shares 60% homology with an unknown protein in Streptomyces sviceus; sfaU2 has a full length of 468 bp, encodes a functionally unknown protein of 155 amino acid residues, which, upon analysis was seen to share 81% homology with a resolvase/integrase in Streptomyces turgidiscabies. A gene sfaU1 knockout was carried out and confirmed that it was not relevant to SFA biosynthesis. Meanwhile, the transposon sequences up- and down-stream of sfaU1 also imply the boundaries of the horizontal transfer of the gene cluster.

3.2 Study of the Right Boundary

Study of the right boundary was focused on sfaV1, sfaV2, sfaV3, and sfaT2. sfaV1 has a full length of 537 bp, it encodes an unknown protein of 178 amino acid residues, and shares 81% homology with an unknown protein in Streptomyces cattleya; sfaV2 has a full length of 459 bp, it encodes a functionally unknown protein of 152 amino acid residues, and shares 46% homology with an functionally unknown protein in Streptomyces coelicolor, sfaV3 has a full length of 435 bp, it encodes an unknown protein of 144 amino acid residues, shares 45% homology with an unknown protein in S. coelicolor, and a certain homology with UDP-N-acetylglucosamine transferase in Pseudomonas syringae pv. tomato. SfaT2 has a full length of 534 bp, encodes a transposase of 177 amino acid residues, and is partly homologous to the IS-4 insertion sequence in Burkholderia vietnamiensis. Based on analysis of the transposon sequence, the downstream boundary was determined to locate adjacent to the transposon region.

Example 4 Proposal for Biosynthesis of the SFA Starter Unit

Endogenous acetoacetyl-CoA is reduced to hydroxy-butyryl CoA by the short chain dehydrogenase/reductases encoded by sfaM, then to Crotonyl-coA by an unknown primary metabolic dehydratase. Then, the crotonyl-coA is reductively carboxylated by crotonyl-coA reductase encoded by sfaR, and amidated by asparagine synthetase analogue encoded by sfaP. Afterwards, the extension by incorporation of the subsequent two-carbon unit is completed via the self-acylation of the starter acyl carrier protein (encoded by sfaE-ACP1). Meanwhile, a free acyltransferase having a modified function and encoded by sfaL is thought to be responsible for hydrolyzing other units which are erroneously added by self-acylation.

Example 5 Proposal for Biosynthesis of the m-Tyrosine in SFA

Traditional phenylalanine/tyrosine/tryptophan hydroxylation systems all rely on an electrophilic substitution reaction to realize hydroxylation in the electron-rich regions of aromatic systems. On the contrary, the meta-hydroxylation of phenylalanine occurs at the relatively electron-poor meta position of phenylalanine. Bioinformatics analysis of the second adenylation domain (A2) in the NRPS shows that its active fingerprint region shares no homology with the fingerprint regions of the adenylation domains which are responsible for recognizing phenylalanine in other systems, and it has no apparent association with tyrosine. The putative meta-hydroxylase, SfaA, was expressed in E. coli and isolated using standard methods. In vitro study of this protein in an AMP PPi assay (for methods see Garneau et al., 2005) showed that it had no activity on free phenylalanine, but did show activity to an acetylcysteamine (SNAC) thioester derivative. This suggests that phenylalanine is the optimal substrate for integration into the polypeptide backbone, i.e. after integration, in situ modification takes place to obtain the meta-tyrosine unit.

Example 6 Proposal for Biosynthesis of the SFA Piperazic Acid Unit

The third building block of the short peptide moiety in the SFA structure is a piperazic acid unit, which is involved in the integration of the peptide backbone in a 2,3-regioselective manner. The piperazic acid unit was initially presumed to be obtained from a proline precursor via dehydrogenation at 1,2-position, ammonolysis, N⁵ oxidation to form cycle, and then reduction. However, previous labeled assays have showed that the real precursor of piperazic acid unit is glutamic acid (Umezawa et al., 2001, Miller et al., 2007). Therefore it is suggested that the piperazic acid unit is formed from a precursor molecule glutamic acid/glutamine via hydrogenation and dehydration, N⁵ oxidation and then hydrogenation. Although no direct evidence of ornithine being the precursor has been obtained in other labeled assay systems, the inventors consider it possible that ornithine is involved in the piperazic acid formation in the system by direct N⁵ oxidation to form the cycle, as seen in other systems (Fujimori et al., 2007).

Example 7 Proposal for Biosynthesis of the Unusual Extension Unit

The iterative polyketide synthase encoded by sfaK is thought to be involved in biosynthesis of the 6-carbon unusual extender unit. Acetyl CoA or malonyl-CoA is used as a starter unit to go through two rounds of two-carbon extension, and triketo hexanoyl CoA is obtained. The keto group in the middle is then selectively reduced by the unusual oxidation-reduction domain at the terminal end of the said enzyme, and a partially reduced CoA derivative with a conjugated double bond is obtained. Then via reductive carboxylation by the crotonyl-coA reductase encoded by sfaR, it is activated to incorporate into the process of polyketide chain extension. In the identification of the fermentation product derived from the knockout mutant of sfaK, the inventors detected a signal of m/z 1033.4 (FIG. 6C). This supports the putative generation of a compensatory biosynthetic product, i.e. the special extension unit side chain is replaced by the substrate methylmalonyl-CoA which is nonoptimal in terms of the corresponding domain for transacylation in the PKS, so that a substitution product with a methyl side chain is obtained.

Example 8 Generation of Non-Natural Analogues of SFA by Genetic Means

The segments corresponding to various DH domains in the SFA biosynthetic gene cluster were inactivated by site-directed mutagenesis, so that a dehydratase lacking active-site residues is encoded. This dehydratase was used to act on an intermediate product which had been through the upstream AT recognition, transfer onto the ACP, condensation catalyzed by the KS, and reduction by the KR. Since the said dehydratase cannot exert its dehydration function to form a double bond, the SFA analogue with a hydroxy group is therefore obtained. The dehydratase domain (DH), is an enzymatic domain which catalyzes the dehydration of the hydroxy group derived from keto reduction, to form a double bond. The functional domain has a highly conserved motif LXXHXXXGXXXXP, wherein the histidine residue is the catalytic active center; leucine and glycine maintain normal folding; proline is sometimes not conserved and is functionally unknown. Thus, the histidine residue was chosen as the target for mutation. DH3/DH4/DH5/DH6 (the DH domains of modules 7, 8, 10 and 11) in SfaA4 (sfaH) (responsible respectively for the formation of the two sets of two double bonds inside and outside of the ester group of the macrolide) were chosen as target functional domains to be inactivated by site-directed mutagenesis. The polymerase chain reaction (PCR) was used to amplify 8 DNA fragments of around 1 kb flanking the motifs of the above-said functional domains, and these fragments were used as homologous arms. The histidine residue was changed into a nonpolar amino acid residue such as glycine/alanine or the like, to remove the dehydration function. Altered codons were introduced by primers, and two fragments carrying the mutation were ligated (restriction sites required for such ligation may be introduced by silent mutation). In this manner, homologous recombinant plasmids needed for site-directed mutagenesis were constructed. Corresponding mutants were fermented. Liquid chromatography/mass spectrometry detected M+18 signal peaks (LC-ESI-MS m/z1109.3, 1131.5), showing that the desired compounds were produced.

To facilitate the understanding of the present invention, Examples are further provided in the following, for illustrative purpose only and not limiting the scope of the invention.

Example 9 Construction of a Genetic Transfer System for the SFA Producing Strain Streptomyces sp A92-309110 (S. Flaveolus)

The target plasmid to be used in conjugative transfer was first transformed in E. coli S17-1. A single colony from a plate was picked and inoculated into tubes with 3 mL LB medium and corresponding antibiotic for selective pressure, and incubated overnight at 37° C. The culture was harvested the next day, 1 mL culture was pipetted and inoculated into a 250 mL shake flask containing 50 mL LB medium and corresponding antibiotic for selective pressure. Incubation was performed at 37° C. in a shaker until the OD₆₀₀ value reached around 0.5. The culture was transferred to 50 mL EP tubes, and centrifuged at 3800 rpm for 10 min at 16° C. Bacteria were recovered, and washed twice with 20 mL LB medium, then centrifuged under the same conditions. Bacteria were then recovered and resuspended in 1 mL LB before use.

One tube of cryopreserved spore suspension was taken and centrifuged at 12000 rpm for 3 min at room temperature. After the supernatant was removed, it was washed twice with 1 mL TES buffer, and resuspended in 500 μL TES buffer. Heat shock was performed in a water bath at 50° C. for 10 min, and then 500 μL TSB medium was added. The solution was well-mixed, incubated at 37° C. for 4-5 h, and then centrifuged to remove the supernatant entirely. The pellet was then resuspended with 1.5 mL LB before use.

The recipient bacteria and donor bacteria (each 100 μL) were spread on plates with MS medium (containing 10 mM MgCl₂), and cultured at 30° C. for 12-16 h. In addition, one plate, which was spread with recipient bacteria only, was used as negative control. After culturing, 4-5 mL sterile ddH₂O was added to each plate, and the surface was gently scraped with a scraper. After the water was absorbed, the plate was dried for 1 h, then again coated with 1 mL sterile ddH₂O (containing 12.5 μg/mL Am and 50 μg/mL NA) and cultured at 30° C. for 3-5 d.

A single well-grown colony which was successfully transformed was picked and inoculated into 3 mL TSB medium, containing antibiotic selection, if necessary. The culture was shaken at 30° C. After 1 d, around 1 μL of the culture was taken out and spread onto a 150 mm plate containing AmR ISP-4, then cultured for 2 d at 37° C. The plate was directly overlaid with solid plate medium containing kanamycin ISP-4, and cultured for 6-7 h at 37° C. Then the 2 pieces of culture medium were separated, and cultured for another 2 d at 37° C. respectively. Single colonies which had grown on AmR but not at the corresponding position on kanamycin plates were picked and inoculated into 3 mL TSB medium. The culture was shaken and ready for fermentation.

Example 10 Construction of a Gene Library of the SFA Producing Strain Streptomyces sp A92-309110 (S. Flaveolus)

10.1 Small-Scale Enzyme Digestion Assay

Firstly, the amount of Sau3Al to use was determined by a series of dilution experiments. A reaction system of 250 μl (containing genomic DNA (eg gDNAQXD01-82-1) 40 μl, BSA (100×) 2.5 μl) was first prepared, then the 250 μl reaction system was divided to 1×50 μl and 7×25 μl, and kept on ice. Then 2 μl of Sau3Al, which was previously diluted to 0.5 u/μl, was added to tube #1 containing the 50 μl reaction system, and well-mixed. Then 25 μl was transferred to the 25 μl in tube #2. These transfer steps were repeated 7 times. All systems were incubated in water bath at 37° C. for 15 min, then inactivated at 70° C. for 10 min. 0.4% agarose gel electrophoresis was run at 4° C. in a cold room, then stained with ethidium bromide. The quality of enzymatic digestion was examined under UV light or using a gel image system.

10.2 Large-Scale Enzyme Digestion Assay

According to the reaction conditions determined in preliminary experiments, 4 times the amount of the total DNA and the enzyme at corresponding concentration were used to prepare DNA fragments needed for constructing the library: The system was well-mixed on ice, divided evenly to 5 aliquots, and incubated in a water bath at 37° C. At 12, 14, 16, 18 and 20 min respectively, aliquots were taken out and inactivated at 70° C. for 10 min. 0.4% agarose gel electrophoresis was run at 4° C. in cold room, then stained with ethidium bromide. The quality of enzymatic digestion was examined under UV light or using a gel image system.

10.3 Dephosphorylation

Digested DNA fragments of suitable size were extracted sequentially with saturated phenol, and chloroform-isoamyl alcohol solution. 0.1 volume of 3M NaAc and 3 volumes of anhydrous alcohol were added to precipitate the DNA. Then the DNA pellet was washed with 70% alcohol, dried, and then resuspended in 200 μl water. 10 μl was removed as a control, and the remaining DNA was dephosphorylated. 10 μl SAP (Promega, 1 unit/μl) and 25 μl 10× buffer were added to 190 μl Sau3Al digested DNA, and then water was added to 250 μl. The system was incubated for 1 h at 37° C., supplemented with 7 μl SAP, and then well-mixed. After 1 h incubation, heat denaturation was performed at 70° C. to inactivate the enzyme. Extraction was performed sequentially with saturated phenol, chloroform:isoamyl alcohol solution. 0.1 volume of 3M NaAc and 3 volumes of anhydrous alcohol were added to precipitate DNA. Then the DNA was washed with 70% alcohol, dried, resuspended in 15 μl TE solution. Then it was examined to see whether dephosphorylation was complete. 0.3% Agarose Gel Electrophoresis was run at 4° C. in a cold room, and the gel was stained with ethidium bromide. The quality of enzyme digestion was examined under UV light or using a gel image system.

10.4 Preparation of SuperCos1 Vector

The single enzyme XbaI was used to linearize SuperCos1 between two cos sites, and then dephosphorylation was performed (to prevent self-ligation). Then, in order to insert partially digested fragments of genome DNA, digestion was performed with BamHI to obtain two arms of 1 kb and 7 kb. The prepared vector had the concentration of 1 μg/μl.

10.5 Ligation of Fragments Derived from Total DNA into SuperCos1 Vector

6 μl dephosphorylated DNA (around 2 μg/μl) and 4 μl prepared SuperCos1 ((around 1 μg/μl)) were mixed, and 1.2 μl was taken out as a control. To the remaining 8.8 μl of the mixture solution were added 1.2 μl T4 DNA ligase (NEB 400 u/μl) and 1.2 μl buffer, then well-mixed. Ligation was carried out for 16 h at 16° C. Agarose Gel Electrophoresis was run at 4° C. in a cold room. The gel was stained with ethidium bromide, and examined under UV light or using a gel image system.

10.6 Library Packaging

One Stratagene Gig Pack III XL packaging reagent was removed from the −80° C. freezer, and quick-thawed by hand. 4 μl ligation product was added, and gently pipetted 3 times. The mixture was incubated in a water bath at 22° C. for 2 hr, then removed and 500 μl SM buffer added, then inverted several times. 50 μl chloroform was added and the mixture inverted several times (now some protein-like precipitation should be visible). The mixture was then centrifuged for a few seconds. The supernatant was transferred by pipetting, and stored at 4° C.

10.7 Titration

Firstly, a single bacterial colony of E. coli VCS257 was picked from a plate and inoculated into LB to culture overnight. 500 μl of culture was taken and inoculated into 50 ml LB (10 mM MgSO₄, 0.2% maltose), and cultured at 37° C. with shaking. When the OD₆₀₀ of E. coli reached 0.84, 5 μl packaging solution was added to 45 μl SM buffer, then added into 50 μl of the E. coli VCS257. The tube was gently tapped to mix, then put into a water bath at 22° C. to incubate for 30 min. 400 μl LB was added, the tube inverted several times, then incubated in a water bath at 37° C. for 75 min (and inverted several times every 15 min). 250 μl was spread onto each LB plate (Amp100 μg/ml), and incubated overnight at 37° C.

10.8 Confirmation of Authenticity of Library

To prove that the bacterial colonies grown were not false-positive but really contained recombinant cosmids, 10 colonies were randomly picked, inoculated into LB (Amp100 μg/ml) and cultured. Cosmids were extracted according to the alkaline lysis method used for E. coli plasmid DNA minipreps, then digested with BamHI and run on a 0.5% Agarose Gel.

10.9 Library Amplification

E. coli VCS257 was inoculated into 50 ml LB (containing 10 mM MgSO₄, 0.2% maltose) and cultured to OD₆₀₀=0.84, then transfection was immediately performed. Cell culture (100 μl) and packaging solution (100 μl) were gently mixed, incubated in a water bath at 22° C. for 30 min, then 800 μl LB was added to each of 5 microcentrifuge tubes, gently mixed, and incubated in a water bath at 37° C. for 75 min with gently mixing every 15 min. Whilst the incubation was being performed, 5 large plates (Amp concentration of 100 μg/ml) were placed at 37° C. in an incubator to prewarm. After incubation in the water bath was finished, plates were spread with the contents of one microcentrifuge tube for each plate, and incubated overnight at 37° C. After culturing for 18 hr (until bacterial colonies had grown well), 3˜4 ml LB were added to each plate. Bacterial colonies were scraped with a scraper, and the culture solution was pipetted and transferred to a 50 ml centrifuge tube. Culture solutions from 5 large plates were pooled, and Ampicillin and sterile glycerol were added to final concentrations of 50 μg/ml and 18% (V/V). 250 μl/tube aliquots were stored at −80° C.

Example 11 Generation of an Engineered Streptomyces sp A92-309110 (S. Flaveolus) Strain Resulting in a Gene Replacement of the sfaK Gene by PCR-Targeting

Generation of an engineered Streptomyces sp A92-309110 (S. flaveolus) strain with an in-frame deletion in the sfaK gene of the sanglifehrin biosynthetic cluster by PCR-targeting involves the majority of the sfaK coding region being replaced by an apramycin resistance marker and the oriT using the standard procedure of PCR-targeting (Gust et al 2002).

Primers 201-1L (SEQ ID NO: 30) and 201-1R (SEQ ID NO: 31) were designed according to the PCR-targeting procedure to amplify the apramycin resistance marker along with the oriT from the template pIJ773 (GenBank accession no. AX657066.1). The 5′ region of each primer (not underlined) is identical to sfaK such that replacement of the sfaK sequence between the binding regions of the oligos with the replacement cassette from pIJ773, containing FRT, oriT, aac(3)IV and FRT will remove the activity of SfaK. The underlined 3′ region of each oligo is identical to a sequence in pIJ773 for amplification of the replacement cassette. Within the replacement cassette, FRT is the FLP-recombinase recognition target sequence and the two FRT sequences flank the origin of transfer from RK2 (oriT) and the apramycin resistance gene (aac(3)IV), one skilled in the art will be familiar with this technique and it is described in detail in Gust et al 2002 and further supported by references within.

201-1L (SEQ ID NO: 30): 5′-CTCGACCGGTACTGGGCCAACGTGGTGGCCGGTGTCGACATTCCGG GGATCCGTCGACC-3′ 201-1R (SEQ ID NO: 31): 5′-GGCCAGTTCGCGCAGGAAGGCCCGTACGCCGTCGTCCGGTGTAGGC TGGAGCTGCTTC-3′

Amplification of the ˜1.4 kb PCR-targeting DNA cassette was achieved using the primers 201-1L (SEQ ID NO: 26) and 201-1R (SEQ ID NO: 27), pIJ773 as the template and Primer Star Polymerase (Takara Co. Ltd.) using standard conditions with an annealing temperature of 50° C. The cosmid pTL3102 covers an appropriate region of the sanglifehrin cluster and was first transformed into E. coli BW25113/pIJ790 to give the strain E. coli BW25113/pIJ790/3102. E. coli BW25113/pIJ790 is maintained at 30° C. in LB (Luria-Bertani medium; Sambrook et al., 1998) containing chloramphenicol (25 μg/mL). To make the gene replacement construct pTL3122 the steps outlined below were carried out. PCR-targeting was effected by transforming the gel purified DNA cassette into E. coli BW25113/3102 by electroporation, after electroporation the bacteria were incubated in LB at 37 degree centigrade for 1 h to induce recombination and express antibiotic resistance, then plated on LB agar containing 100 μg/mL apramycin for overnight incubation at same temperature. Apramycin resistant colonies were inoculated into LB supplemented with 100 μg/mL apramycin and incubate for overnight at 37° C. Cosmid DNA was isolated, and the presence of the resistance cassette confirmed by restriction enzyme digestion and DNA sequence analysis, the cosmid generated was named pTL3111. To facilitate transformation into Streptomyces, the cosmid size was reduced by cutting pTL3111 with BglII. The 13 kb BglII fragment containing the inactivated sfaK and acc(3)IV-containing resistance cassette was subcloned into pKC5201 to give the final replacement construct pTK3122. pKC5201 was derived from pKC1139 by replace the acc(3)IV with the neomycin resistance gene of supercos1. To generate the sfaK replacement mutant in Streptomyces sp A92-309110 (S. flaveolus), pTL3122 was transformed into E. coli S17-1 by electroporation to generate the E. coli donor strain for conjugation. Streptomyces sp A92-309110 (S. flaveolus) was transformed by conjugation with E. coli 517-1/pTL3122 as described in the general methods above. Apramycin resistant ex-conjugants were homogenized and streaked onto the ISP-4 agar supplemented with 50 μg/mL apramycin and cultured at 37° C. until well grown colonies were achieved. A series of single clones were used to inoculate 3 mL TSB broth supplemented with 30 μg/mL of apramycin shaking at 250 rpm, 37° C. for 4 days. 100 μL of each culture was spread onto ISP-4 agar an incubated at 37° C. for 2-3 days to induce the second crossover event. Single colonies were plated +/−neomycin to determine loss of the plasmid backbone. Neomycin sensitivity indicated loss of the plasmid sequence and colonies with that phenotype were analysed by PCR to confirm the replacement of sfaK with the DNA cassette had been achieved. One such colony was designated Streptomyces flaveolus mv. xuwengensis, with the alternative name Streptomyces sp. TL3011. Strains were then cultured according to the methods described, and the strains were seen to make Sanglifehrin X (see FIG. 6C).

Example 12 Generation of Engineered Streptomyces sp A92-309110 (S. Flaveolus) Strains Resulting in Gene Replacements of a Series of Sanglifehrin Biosynthesis Genes by PCR-Targeting

Using similar methods to that described in example 11 above, replacement mutants of sfaA, sfaB, sfaC, sfaJ, sfaL, sfaM, sfaN and sfaP were constructed. In each case, majority of the coding region of the target gene was replaced by an apramycin resistance marker and the oriT using the standard procedure of PCR-targeting (Gust et al 2002). A pair of primers was designed for each target gene (Table 2) according to the PCR-targeting procedure to amplify the apramycin resistance marker along with the oriT from the template pIJ773 (GenBank accession no. AX657066.1). The 5′ region of each primer (not underlined) is identical to the target gene such that replacement of the gene sequence between the binding regions of the oligos with the replacement cassette from pIJ773, containing FRT, oriT, aac(3)IV and FRT will remove the activity encoded by the gene. The underlined 3′ region of each oligo is identical to a sequence in pIJ773 for amplification of the replacement cassette. Within the replacement cassette, FRT is the FLP-recombinase recognition target sequence and the two FRT sequences flank the origin of transfer from RK2 (oriT) and the apramycin resistance gene (aac(3)IV), one skilled in the art will be familiar with this technique and it is described in detail in Gust et al 2002 and further supported by references within.

Amplification of the ˜1.4 kb PCR-targeting DNA cassette was achieved using the primers shown in table 2, pIJ773 as the template and Primer Star Polymerase (Takara Co. Ltd.) using standard conditions.

TABLE 2 Primers used for PCR-Targeting Target Primers Primer sequences gene 201-2L SEQ ID NO: 32 GTGGAAATCGGCTCGGGCGCGCCC sfaA GAATTAACCGCGTCGATTCCGGGGA TCCGTCGACC 201-2R SEQ ID NO: 33 AATGGATGTATCGTCGCAGGACGCC CAGAATTCACCTGCTGTAGGCTGGA GCTGCTTC 201-3L SEQ ID NO: 34 GCGCAGCAGAGCCCGGAATCAGAA sfaB GTACTGGACGTCACCATTCCGGGGA TCCGTCGACC 201-3R SEQ ID NO: 35 GGCGATCTCGCCCGCGCGGACCGC CACCATGGACAGCAGTGTAGGCTGG AGCTGCTTC 201-4L SEQ ID NO: 36 GAGGATTGCGACGGCGTCGTCCTG sfaC GCGTTTCTGCGACACATTCCGGGGA TCCGTCGACC 201-4R SEQ ID NO: 37 CTCCTCGTCGGCTTCGGTGAGTCCG CGGTCGCGCATCACTGTAGGCTGGA GCTGCTTC 201-5L SEQ ID NO: 38 GGGCCGCAGGACAGGTCCGGCGGC sfaJ CCGGTGCGCGGCGAGATTCCGGGG ATCCGTCGACC 201-5R SEQ ID NO: 39 CGCGCCGGAGAACAGCGGGAAGTA GGTGTCGAGGTCGTCTGTAGGCTGG AGCTGCTTC 201-6L SEQ ID NO: 40 GGCCGGCCCCGGAGCCCTGGGCGC sfaL CGCCCTGCGTTCGGGATTCCGGGGA TCCGTCGACC 201-6R SEQ ID NO: 41 GCTCTCCACGGAGGCGCTCACCGC GGCGACGGCGGCCTCTGTAGGCTG GAGCTGCTTC 201-7L SEQ ID NO: 42 GCGGTCGTGACCGGATCGTCCCGC sfaM GGCATCGGCGCGGCCATTCCGGGG ATCCGTCGACC 201-7R SEQ ID NO: 43 CACGCCGTCGGCGGTCCAGCCGCC GTCGAAGCGCAGGGTTGTAGGCTG GAGCTGCTTC 201-8L SEQ ID NO: 44 CTTCGGCGTCCTCGCGCTCGCCCAC sfaN GCCCTCGGCGATCCATTCCGGGGAT CCGTCGACC 201-8R SEQ ID NO: 45 GTGCATGCCGATGGACAGGCCCGC GAGCGCGACCACGTCTGTAGGCTG GAGCTGCTTC 201-9L SEQ ID NO: 46 GACGACCTCGCGCGGCACCGGTCC sfaP GTCGTCCAGGCGATGATTCCGGGGA TCCGTCGACC 201-9R SEQ ID NO: 47 CATCTCGATGCCGCCCCGGTCGTGC GGCAGGCTGAAGTCTGTAGGCTGGA GCTGCTTC

To make the gene replacement constructs, PCR-targeting is executed by transforming the gel purified cassette fragment into E. coli Bw25113/pIJ790/3106 (for sfaA, sfaB and sfaC as shown in table 3 or E. coli Bw25113/3102 (for sfaJ, sfaL, sfaM, sfaN and sfaP as shown in table 3) by electroporation. The strains are made in advance by transforming the cosmid pTL3106 or pTL3102 into E. coli Bw25113/pIJ709 by electroporation. After transformation, the bacteria was incubated in LB at 37° C. for 1 h to induce recombination and express antibiotic resistance, and then plated on LB agar containing 100 μg/mL of ampramycin for overnight incubation at 37° C. Apramycin resistant colonies were used to inoculate LB supplemented with 100 μg/mL ampramycin and incubated for overnight at 37° C. The recombinant cosmids are isolated by plasmid isolation Kit (Dingguo Co. Ltd.) and confirmed by restriction enzymes digestion and DNA sequence and given then names shown in table 3. Due to the low transformation efficiency for larger cosmid DNA into the recipient Streptomyces sp., direct transformation of the recombinant cosmids into streptomyces by conjugation were only successful for pTL3113 and pTL3114 which contained the cassettes for inactivation of sfaB and sfaC and resulted in the mutant strains TL3003 and TL3004 respectively. For the remainder, the inserts were shortened size by cutting the recombinant cosmids with restriction enzymes as shown in table 3. The resulting DNA fragments were subcloned into pKC5201 which is derived from pKC1139 by replace the acc(3)IV with the neomycin resistance gene of supercos1 to give the plasmids listed in table 3. These plasmids resulted in the incorporation of the desired mutations and the mutant strains were given the designated names shown in table 3; such that strain Streptomyces sp. TL3002 has sfaA replaced by the acc(3)IV-containing resistance cassette, Streptomyces sp. TL3005 has sfaJ replaced by the acc(3)IV-containing resistance cassette, Streptomyces sp. TL3006 has sfaL replaced by the acc(3)IV-containing resistance cassette, Streptomyces sp. TL3007 has sfaM replaced by the acc(3)IV-containing resistance cassette, Streptomyces sp. TL3008 has sfaN replaced by the acc(3)IV-containing resistance cassette and Streptomyces sp. TL3009 has sfaP replaced by the acc(3)IV-containing resistance cassette.

TABLE 3 The recombinant cosmids and plasmids Enzymes to Fragments size Mutant Target Recombinant shorten after digestion Plamid generated gene Cosmid Cosmids the size (kb) name TL3002 sfaA pTL3106 pTL3112 Bg/II 8 pTL3123 TL3003 sfaB pTL3106 pTL3113 N.A. N.A. N.A. TL3004 sfaC pTL3106 pTL3114 N.A. N.A. N.A. TL3005 sfaJ pTL3102 pTL3115 Bg/II/ 11 pTL3124 EcoRI TL3006 sfaL pTL3102 pTL3116 EcoRI 10 pTL3125 TL3007 sfaM pTL3102 pTL3117 EcoRI 11 pTL3126 TL3008 sfaN pTL3102 pTL3118 EcoRI 10.5 pTL3127 TL3009 sfaP pTL3102 pTL3119 EcoRI 9.5 pTL3128

Plasmids or cosmids for transformation of Streptomyces are first transformed into E. coli s17-1 and then conjugated into Streptomyces as described in example 9 above. The apramycin resistant conjugants are homogenised and streaked onto ISP-4 agar media supplemented with 50 μg/ml ampramycin and cultured at 37° C. until well grown colonies were achieved. A series of single clones of each were used to inoculate 3 mL TSB broth supplemented with apramycin 30 μg/mL shaking at 250 rpm at 37° C. for four days. 100 μL of culture was spread onto ISP-4 agar at 37° C. for 2-3 days to induce the second crossover event. Single colonies were plated with and without neomycin to determine loss of the plasmid backbone. Neomycin sensitivity indicated loss of the plasmid sequence and colonies with that phenotype were analysed by PCR to confirm the replacement of the target gene with the DNA cassette had been achieved. The final mutant strains were given the strain names indicated in table 3.

Example 13 Generation of Engineered Streptomyces Sp A92-309110 (S. Flaveolus) Strains Resulting in Gene Deletion of sfaR

Cosmid pTL3102 was digested with BglII and KpnI. The resulting 4.7 kbp DNA fragment was and cloned into BglII/KpnI-digested pSP72 to create pTL3132. An internal 674 bp DNA fragment of pTL3132 was removed by Eco72I digestion and the vector self-ligated to create pTL3133. The BglII and HindIII fragment of pTL3133 was cloned into the BamHI/HindIII-digested pKC1139 to make pTL3129, which was used to engineer gene deletion of sfaR.

The generation of the desired double recombinant strain was carried out using similar procedures to those described above. E. coli 517-1/pTL3129 was used to transform Streptomyces sp A92-309110 (S. flaveolus) by conjugation. The apramycin resistant conjugants were homogenised and streaked onto ISP-4 agar media supplemented with 50 μg/mL ampramycin and cultured at 37° C. until well grown colonies were achieved. A series of single clones of each were used to inoculate 3 mL TSB broth supplemented with apramycin 30 μg/mL shaking at 250 rpm at 37° C. for four days. 100 μL of culture was spread onto ISP-4 agar at 37° C. for 2-3 days to induce the second crossover event. After inducing the double crossover recombination at 37° C. colonies with negative apramycin phenotype are picked out for genotype conformation by PCR. The desired PCR product is about 700 bp different from the 1.4 kbp wild-type pattern. The final strain which was deleted in sfaR was designated Streptomyces sp. TL3010.

Example 14 Generation of DH Mutants in Each of the 4 DH Domains of sfaH

The four DH domains of sfaH are contained within PKS modules 7, 8, 10 and 11. As described in the patent text above, the activity of a DH domain can be removed by site-directed mutagenesis to alter the active site histidine to a nonpolar amino acid. This was carried out for the four DH domains of modules 7, 8, 10 and 11 by amplifying 2 arms for each DH using the primers outlined in table 4 below.

TABLE 4 primers SEQ ID NO sequence DH (module 7) 105A-2 SEQ ID NO: 48 TTT GGATCC TACACC left arm GGCCAGGGCGCCC 105C-2 SEQ ID NO: 49 TTT GGTACC GAGGAC GCTAGCGTCGGCCAG CCAGGGGTGC DH (module 7) 105B-2 SEQ ID NO: 50 TTT AAGCTT AGCACC right arm CGTGCCACCGGTCAC 105D-2 SEQ ID NO: 51 GCTAGCGTCCTC GGT ACC CCGGTGCTCCCC GGCACC DH (module 8) 106A-2 SEQ ID NO:52 TTT GGATCC GGTGTT left arm GTGGGCGGTGATGG 106C-2 SEQ ID NO:53 TTT GAATTC GGCG AG TACT ACGGCATCGGC CGTCCAGGCGGC DH (module 8) 106B-2 SEQ ID NO:54 TTT AAGCTT GCACGA right arm CGTGGCCGAAGCG 106D-2 SEQ ID NO:55 TTT GAATTC GCCGT A GTACT CGCCACCACC CTGCTGCCCGGC DHmod10 left 107A SEQ ID NO: 56 TTT GAATTC GGGTGG arm TCCGGAGCTGGATCG 107C SEQ ID NO: 57 CGGCAGCAGGGCAGG G ACTAGT ATGGCGGC DH (module 10) 107B SEQ ID NO: 58 TTT AAGCTT GGTGCT right arm CGGACTGGGCGGAGC 107D SEQ ID NO: 59 GCCGCCAT ACTAGT C CCTGCCCTGCTGCCG DH (module 11) 108A SEQ ID NO: 60 TTT GAATTC CGACGA left arm CACCGGATACGGGC 108C SEQ ID NO: 61 CGGCAGCAGGACGGT G ACTAGT ACGGTGGC DH (module 11) 108B SEQ ID NO: 62 TTT AAGCTT CCACCC right arm ATGTCTGCACCAGG 108D SEQ ID NO: 63 GCCACCGT ACTAGT C ACCGT

Therefore, in order to generate the appropriate sequence to mutate the DH of module 7, the left arm was amplified with primers 105A-2 and 105C-2 and the resulting 1.2 kbp DNA fragment cloned into pANT841 to give 01-44-1 and the right arm was amplified with primers 105B-2 and 105D-2 and the resulting 1.2 kbp DNA fragment cloned into pANT841 (Genbank: AF438749) to give 01-44-2.

In order to generate the appropriate sequence to mutate the DH of module 8, the left arm was amplified with primers 106A-2 and 106C-2 and the resulting 1 kbp DNA fragment cloned into pANT841 to give 01-44-3 and the right arm was amplified with primers 106B-2 and 106D-2 and the resulting 1 kbp DNA fragment cloned into pANT841 to give 01-44-4.

In order to generate the appropriate sequence to mutate the DH of module 10, the left arm was amplified with primers 107A and 107C and the resulting 1 kbp DNA fragment cloned into pTLV1 to give 01-28-1 and the right arm was amplified with primers 107B and 107D and the resulting 1 kbp DNA fragment cloned into pTLV1 to give 01-28-2.

In order to generate the appropriate sequence to mutate the DH of module 11, the left arm was amplified with primers 108A and 108C and the resulting 1 kbp DNA fragment cloned into pTLV1 to give 01-28-3 and the right arm was amplified with primers 108B and 108D and the resulting 1 kbp DNA fragment cloned into pTLV1 to give 01-28-4.

Construction of the final plasmid for each target mutation, entails three-fragment ligation. The plasmids containing the PCR fragments that carry the target left and right regions of homology were digested with appropriate restriction enzymes and ligated into appropriately digested vector pKC1139 (BamHI/KpnI for 105AC, KpnI/HindIII for 105BD, BamHI/EcoRI for 106AC, EcoRI/HindIII for 106BD, EcoRI/SpeI for 107AC, HindIII/SpeI for 107BD, EcoRI/SpeI for 108AC, HindIII/SpeI for 108BD). Ligation reactions were used to transform E. coli DH5α. Plasmid DNA was analysed by restriction digestions. Final plasmids to effect the double cross-over events for transformation of Streptomyces are first transformed into E. coli s17-1 and then conjugated into Streptomyces as described in Example 9 above The apramycin resistant conjugants were homogenised and streaked onto ISP-4 agar media supplemented with 50 μg/ml ampramycin and cultured at 37° C. until well grown colonies were achieved. A series of single clones of each were used to inoculate 3 mL TSB broth supplemented with apramycin 30 μg/mL shaking at 250 rpm at 37° C. for four days. 100 μL of culture was spread onto ISP-4 agar at 37° C. for 2-3 days to induce the second crossover event. Single colonies were plated +/−apramycin to determine loss of the plasmid backbone. Apramycin sensitivity indicated loss of the plasmid sequence and colonies with that phenotype were analysed by PCR followed by restriction enzyme digestion of the amplified fragment in each case to confirm the mutation of DH domains. Restriction enzyme digestion of the PCR products of these DH mutants gives two fragments due to the restriction site that is introduced along with the mutation, which is contrary to that of the wild type. Strains were grown under standard fermentation conditions described above, and seen to produce the desired products (See FIG. 6).

Example 15 Characterization of SfaR as a Pathway-Specific Reductase/Carboxylase

To validate the central role of SfaR for unusual building block supply in the proposed pathway, we first inactivated sfaR by in-frame deletion. The resultant mutant completely lost the ability to produce SFA, suggesting that it functions as a pathway-specific ccr homolog essential to the SFA biosynthesis (see example 16). Secondly, we heterologously expressed and purified the N-terminal 6× His-tagged SfaR to near homogeneity for in vitro studies. As expected, in the presence of NADPH and bicarbonate, crotonyl-S-CoA (FIG. 7, 4) was efficiently converted by SfaR to the major product ethylmalonyl-S-CoA (FIG. 7, 5) along with the shunt product butyryl-S-CoA (FIG. 7, 7). Omitting bicarbonate in the reaction mixture, crotonyl-S-CoA was accordingly reduced to butyryl-S-CoA. For crotonyl-S-CoA, K_(m)=8.4×10−4 M, K_(cat)=6.8×10−3/min, and K_(m)/K_(cat)=1.2×10−1 M·min. To exclude the possibility that the reaction directly occurs on the ACP, SfaO was expressed and purified as a N-terminal 6×His-tagged derivative. Attempts with crotonyl-S-SfaO as the substrate failed to detect any activity of SfaR, providing the evidence that SfaR carries the activities on the CoA-based crotonyl-S-CoA rather than the ACP-based crotonyl-S-SfaO (FIG. 7, 3). Thirdly, to detect the substrate flexibility of SfaR, we synthesized the 5-carbon substrate pentenyl-S-CoA. Intriguingly, under the conditions with and without bicarbonate, SfaR performed the reduction and reductive carboxylation on pentenyl-S-CoA (FIG. 7, 8) rapidly to generate pentanoyl-S-CoA and propylmalonyl-CoA (FIG. 7, 10), respectively, showing the efficiencies comparable to the activities on the 4-carbon substrate, crotonyl-S-CoA. Under the conditions used, the reductive carboxylation took place too fast to be measured for kinetics analysis; and for pentenyl-S-CoA in reductively producing pentanoyl-S-CoA, K_(m), K_(cat) and K_(m)/K_(cat) values were 1.4×10−3 M, 4.2×10−2/min and =3.4×10−2 M·min, respectively.

Example 16 Generation of Engineered Streptomyces sp A92-309110 (S. Flaveolus) Strains Resulting in Gene Deletion of sfaK and Production of Novel Compounds

PCR is carried out using genomic DNA or cosmid DNA as a template, and the primer pairs sfaK-L-for (SEQ ID: 64) and sfaK-L-rev (SEQ ID: 65) to generate one PCR product (designated SfaK-L), and sfaK-R-for (SEQ ID: 66) and SfaK-R-rev (SEQ ID: 67) to generate the other (designated sfaK-R). These PCR products are isolated, and then digested with restriction enzymes (sfaK-L with EcoRI and BamHI, and sfaK-R with BamHI and HindIII), then ligated into pKC1139 previously digested with EcoRI and HindIII. The plasmid psfaKKO is isolated.

The generation of the desired double recombinant strain is carried out using similar procedures to those described in previous examples. E. coli S17-1/psfaKKO is used to transform Streptomyces sp A92-309110 (S. flaveolus) by conjugation. The apramycin resistant conjugants are homogenised and streaked onto ISP-4 agar media supplemented with 50 μg/mL ampramycin and cultured at 37° C. until well grown colonies are achieved. A series of single clones of each are used to inoculate 3 mL TSB broth supplemented with apramycin 30 μg/mL shaking at 250 rpm at 37° C. for four days. 100 μL of culture is spread onto ISP-4 agar at 37° C. for 2-3 days to induce the second crossover event. After inducing the double crossover recombination at 37° C. colonies with negative apramycin phenotype are picked out for genotype confirmation by PCR. The desired PCR product is about 1700 bp different from the wild-type pattern. The final strain which is deleted in sfaK is designated Streptomyces sp. sfaKKO.

Streptomyces sp. sfaKKO is grown under standard fermentation conditions described above. The fermentation broth is seen to contain a new peak corresponding to a sanglifehrin of molecular weight 1034.3, presumed to be the product sanglifehrin X. Products are isolated using standard methods.

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Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

All patents and patent applications mentioned throughout the specification of the present invention are herein incorporated in their entirety by reference.

The invention embraces all combinations of preferred and more preferred groups and suitable and more suitable groups and embodiments of groups recited above. 

The invention claimed is:
 1. An isolated nucleic acid molecule comprising the sanglifehrin A biosynthesis gene cluster of SEQ ID NO: 1, wherein: (i) sfaK (residues 97396-101943 of SEQ ID NO: 1), encoding a polypeptide of SEQ ID NO: 14, has been deleted or inactivated, whereby the linear polyketide synthase gene sfaK is knocked out; and/or (ii) sfaA (residues 17024-17854 of SEQ ID NO: 1), encoding a polypeptide of SEQ ID NO: 4, has been deleted or inactivated, whereby the phenylalanine hydroxylase gene sfaA is knocked out.
 2. An isolated nucleic acid comprising a hybrid polyketide producing gene cluster based on the sanglifehrin A biosynthesis gene cluster of SEQ ID NO: 1 in which one or more genes have been deleted, mutated so as to make inactive or less active an enzymatic or regulatory function, wherein the genes that have been deleted, mutated so as to make inactive or less active an enzymatic or regulatory function is (i) sfaK (residues 97396-101943 of SEQ ID NO: 1), encoding a polypeptide of SEQ ID NO: 14, whereby the linear polyketide synthase gene sfaK is knocked out; and/or (ii) sfaA (residues 17024-17854 of SEQ ID NO: 1), encoding a polypeptide of SEQ ID NO: 4, whereby the phenylalanine hydroxylase gene sfaA is knocked out.
 3. A hybrid polyketide producing gene cluster nucleic acid according to claim 2 wherein one or more genes have been replaced by (a) a domain, module or gene from elsewhere in the sanglifehrin A biosynthesis gene cluster or (b) a domain, module or gene which is heterologous to the sanglifehrin A biosynthesis gene cluster.
 4. A hybrid polyketide producing gene cluster nucleic acid according to claim 2 wherein one or more domains, modules or genes have been mutated to inactivate or make less active an enzymatic or regulatory function.
 5. An isolated nucleic acid according to claim 2 which is a DNA.
 6. A vector comprising a DNA according to claim 5 together with one or more promoters or other regulatory elements.
 7. A host cell transformed with a vector according to claim
 6. 8. A host cell according to claim 7 transformed with a vector comprising nucleic acid encoding all or part of the sanglifehrin A biosynthesis gene cluster which host cell does not naturally produce sanglifehrin A.
 9. A method for producing a polyketide which comprises culturing a transformed host cell according to claim
 8. 